Identification, selection and use of high curative potential t cell epitopes

ABSTRACT

A method for identifying T-cell epitopes which can be used to elicit T cells targeting cells capable of regenerating cancers is disclosed. The method identifies T-cell epitopes with a high curative potential, high potency and high probability of T cell recognition (HP). The method includes: (i) identifying high curative potential tumor protein target i.e., identifying HP-TP; (ii) identifying peptide sequences within the protein sequence of an HP-TP that have a high probability of eliciting T cell killing; and (iii) qualifying the sequence specificity based on the fold difference between the specific target and non-targets. The identified T-cell epitopes include a core sequence of 9 amino acids homologous to a sequence expressed within a qualified HP-TP. The T-cell epitopes can be used in a method for reprogramming T cells to selectively attack tumor cells capable of perpetuating a tumor and treating patients, for example, cancer patients.

CROSS REFERENCE TO RELATED APPLICATION

This application claims benefit of and priority to U.S. ProvisionalPatent Application No. 62/087,002 filed on Dec. 3, 2014, incorporated byreference in its entirety.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted Dec. 3, 2015 as a text file named“IBT_101_Sequence_Listing.txt”, created on Dec. 3, 2015, and having asize of 102,644 bytes is hereby incorporated by reference pursuant to 37C.F.R. §1.52(e)(5).

FIELD OF THE INVENTION

The invention is generally directed to methods for identifying T-cellepitopes with high curative potential, high potency and high probabilityof T cell recognition, the T-cell epitopes and their use.

BACKGROUND OF THE INVENTION

Targeted antibody technologies have advanced the treatment of cancer.For example, cancer immunotherapies involving antibody-drug targets haveimproved targeted cancer cell killing. Cancer vaccines used to engendera targeted T cell response have met with more limited success. In allcases, the therapies are rarely curative. At least some of the modestefficacy can be attributed to lack of highly effective targets.

Adoptive Cell Transfer (ACT) is one of the most potent approaches tocancer immunotherapy due to its direct enhancement of T cell killing.Recently, the curative potential of ACT has been demonstrated clinicallyin leukemia and melanoma. Tumor infiltrating lymphocytes (TIL) (a sourceof tumor-reactive T cells) have been harvested for ACT, expanded andtransferred back to patients to increase the number of tumor-reactive Tcells. The antigens TIL recognize are unidentified, but presumed to betumor related. This approach has achieved durable regression in somepatients (about 20% of patients on average), but not in the majority ofthose treated. The TIL repertoire can be refined by selectivelyexpanding a T cell population using one or more antigens to stimulatespecific sub-populations of T cells before transfer.

Ideally, a cancer therapy should eliminate a cancer'sregeneration-capable cells (C-RC) to achieve the best possibility fordurable regression. The use of ACT is severely limited for most solidcancers because of the inability to direct enhanced T cell killing tobiologically-relevant tumor markers, i.e., proteins essential to therecurrence of the cancer. These proteins enable the cancer cells tosurvive and regenerate the cancer. ACT that targets a tumor's C-RC isparticularly needed in cancers of vital organs, where complete ablationof a normal, functionally critical cell type is not feasible. ACT isalso one of the most promising options for the treatment of late-stagemetastatic cancers but most likely only if high probability, highpotency, high specificity T-cell antigens can be identified withinproteins essential to regenerative capacity. While the use if TILincreases the opportunity for relevant tumor reactivity, its ultimateeffectiveness is limited by lack of peptide antigens (Ag) with highcurative potential, high potency and high probability (collectivelydenoted as “HP”) of T cell recognition (HP-Ag).

Tumor-reactive TIL may be used to discover antigenic tumor proteintargets. However, this is laborious and the TIL approach to targetdiscovery has several drawbacks that limit the discovery of HP-Ag.Methods that rely on a patients' immune response to identify T cellepitopes can be highly individualized and can miss many potentiallyvaluable antigens. In many patients the immune system has gone throughcountless refinements and insults leading to a skewed, less than optimaland often ineffective response. Inherent selection of antigen by anindividual's immune system is a major drawback to the development of HPACT (HP-ACT) because of its bias towards certain antigens that may benaturally dominant but not useful for killing the C-RC.

The presence of reactive TILs in patients that have advanced cancerindicates that mere T cell recognition within the tumor is not enough.Aside from supporting the immune response with T cell checkpointblockade or the use of interleukins, there must be an adequate number ofT cells within the tumor or in the circulation. While this is somethingthat ACT can achieve, for it to be an HP-ACT therapy, at least some ofthese T cells must respond to at least one peptide antigen that ispivotal to the C-RC phenotype.

The fact that targeting a pivotal protein essential for perpetuation ofthe cancer is the way to achieve a reliable, durable response in solidtumors has gone unrecognized. The lead author of a recent detailedgenomic analysis on the “non-Darwinian evolution” of a tumor'smutational landscape concluded that a cancer should be nearly impossibleto eliminate with a single target due to extremely high geneticdiversity (Ling et al., Proc Nat Acad Sci USA., 112(47):E6496 (2015)).However what has gone unappreciated is that, while this is true for mostmutations, it will not be true for proteins essential to regeneration ofthe cancer, i.e., those pivotal to the C-RC. Irrespective of the complexand differing mutational landscape in each individual, there areproteins pivotal to perpetuation and the C-RC that are likely to beshared by genetic subtypes of cancer. If one can target those pivotalproteins involved in key pathways that are needed for the type of cancerto persist and use it an effective modality like ACT, then it createsthe opportunity to eliminate the cancer using a single or a minimalnumber of targets. There are additional practical advantages totargeting a protein responsible for a key oncogenic pathway: it meansthat expression of the protein is more likely to be one that persists asthe tumor progresses and metastasizes. This is evidenced in theexpression of at least two HP-TP proteins (AKAP4 and TMPRSS2-ERG)described herein, In addition, if the C-RC driver is lost due tomutation, the likelihood is that those cancer cells will have evolvedinto something less lethal, if they survive at all. Using ACT as themodality targeted to the C-RC will eliminate the cancer before it has anopportunity to develop resistant/alternative clones as seen in responseto targeted drugs or immune therapies that leave the C-RC behind.Therefore the combination of a C-RC target and the modality deliver thetherapy's high curative potential. Methods used to discover epitopes aspresented in antigen presenting cells (APC), such as dendritic cells,fail to fully consider the connective steps required to move an immuneresponse from APC and antigen digest to presentation and activation ofeffector T cells. In many patients these steps are flooded withirregularities from previous treatments and immune regulators leading toa lower probability of epitope effectiveness. These methods do notevaluate the value of the protein associated with the target up frontleading to a large amount of work for data that may be of low curativevalue. Solely genomic methodologies do not necessarily capture the exomeand may be limited by pre- and post-transcriptional regulation, makingepitope evaluation of little translatable value without substantialfurther investigation. Strictly screening stem cell exomes, eithergenomic or proteomic, limits targets to normal developmental orproliferative antigens and may miss mutation-, translocation-derived ornovel expressed antigens, Moreover, most proliferative or metabolicantigens are likely conserved and in use in normal tissue turnover.

Genomic screens with limited additional expression patterning analysiscan lead to simple overexpression candidates. This is exemplified by thestudies of Ochsenreither, et al. (Ochsenreither, et al. Blood119(23):5492-5501 (2012)) where, after a large effort, Cyclin-A1presented as a viable target, yet, the normal expression pattern ofCyclin-A1 makes it a poor target, highly susceptible to off-targetresponses or possibly normal immune regulatory diminution of theresponse. Multiplatform analyses based on primarily genomic (Hoadley, etal. Cell 158(4):929-944 (2014)) data have been performed with relativelypredictable results uncovering genetic mutations and amplificationsclustered in well-known pathways such as p53 and PI3kinase within thesubtypes these categorize. There remains a need for methods foridentifying T cell epitopes that target cells capable of regeneratingcancers, and hence have curative potential.

It is therefore an object of the present invention to provide a methodfor identifying T-cell epitopes which target cells capable ofregenerating cancers.

It is also an object of the present invention to provide epitopes with ahigh curative potential, high potency and high probability of T cellrecognition.

It is still an object of the present invention to provide methods andsystems for programming T cells to selectively attack important tumorcells involved in proliferation, or invasion in an individual.

SUMMARY OF THE INVENTION

A method for identifying T-cell epitopes which target cells capable ofregenerating cancers (“C-RCs”) is disclosed. The method identifiesT-cell epitopes with a high curative potential i.e. durable eliminationof the cancer. The high curative potential is afforded by: 1) acalculated probability of T cell recognition based on multiplebiochemical parameters of antigen interaction that collectively are asgood or better than known positive T cell antigens; and 2) a highpotency afforded by: a) a requirement that the target cancer proteinplay an essential role in the perpetuation of the cancer type and stage;and b) stringent specificity of the peptide antigen that allowsaggressive treatment with little or no on- or off-target T-cellactivation and killing beyond the tumor (HP). The method includes: (i)identifying high curative potential target proteins (HP-TP) i.e.,identifying HP-TP; (ii) identifying peptide sequences within the proteinsequence of an HP-TP that have a high probability of eliciting T cellkilling; and (iii) qualifying the sequence specificity based on the folddifference between the specific target and non-targets.

The method of step 1, identifies a HP-TP based on: 1) its pattern ofcancer expression, number of patients and their accessibility, and itsclinical and commercial feasibility (collectively, parameters ofFrequency); 2) its ability to discriminate cancer cells from normalcells (Specificity); and 3) the strength of its functional relationshipto the cancer's ability to perpetuate itself (Functional Connectivity).These characteristics either contribute or detract from the value of theTP (target protein) as an HP-TP. A TP must have a positive value in allthree parameters to move to Step 2.

Frequency values are calculated based on whether the TP is expressed inmultiple cancers, a specific type of cancer of single origin, or ashared phenotype arising from multiple origins. Then the TP is gradedwithin the category based on the frequency of expression and the numberof advanced diagnoses for the cancer target(s). The frequencycalculation determines how difficult the TP will be to test clinicallyand pursue commercially based on: number of patients, how difficult itwill be to identify those patients and perform clinical studies, and theability to leverage a target across multiple cancers. For example, TPsthat are highly individualized with only a very small percentage ofpatients expressing the protein (for example less than 10 percent)within a large patient population that cannot be further defined basedon other characteristics, for example, lifestyle, medical history,genetic profile, tumor morphology, protein expression, etc., will havezero or negative frequency value because the TP will be impractical toscreen for, test clinically and therefore ultimately treat. A TP that isinfrequent within a population or expressed in very rare cancers maystill be feasible if the TP is also applicable to other cancers and thepatient population can be readily identified, preferably without addedcost. Therefore, the Frequency value of a TP is based on its overallpositive value that encompasses all applications. Commercial feasibilityis a practical need required to achieve production and delivery of thetherapy to a widespread patient population. A TP must have a positivefrequency score to proceed to Step 2.

Specificity is valued based on normal expression, the novelty of adultexpression based on its being a neoantigen due to mutation orrearrangement, a re-expressed developmental protein, or a protein withnovel adult expression, such as certain cancer testis antigens normallyconfined to the testis. Normal expression and the extent of thisexpression will contribute negative values whereas a neoantigen causedby a chromosomal rearrangement expressed only in cancer will contributea positive value. The overall specificity score of the TP must bepositive to continue to Step 2.

Functional Connectivity is valued based on the scientific evidence thatis available to connect the proteins function to a function pivotal tothe perpetuation of the cancer, where without its expression, the cancercell is unlikely to have regenerative capacity. Science thatspecifically demonstrates that the protein is involved in developmentalprocesses or other stem cell biology adds positive value. TPs involvedin pathways that are enabling (like assisting migration for example) butnot pivotal to the survival and perpetuation of the cancer, are notassigned any positive value for this parameter. A TP must have apositive functional connectivity value to qualify as an HP-TP. However,TP determined to be involved in a non-pivotal, i.e., auxiliary functioncan proceed to Step 2 as an Aux-TP if the TP has positive Frequency andSpecificity values. Candidate TP that have insufficient scientificinformation to score its functional connectivity are put on holdawaiting additional information.

Also provided are T-cell antigens with a high curative potential, highpotency and high probability of T cell recognition as not all parts of aTP will be antigenic. The T-cell antigens include a core sequence ofnine amino acids homologous to a sequence expressed within a qualifiedHP-TP; 2) a calculated high probability of T cell recognition andresponse (determined using an integrated comprehensive algorithm or acurated combination of algorithms); 3) a high degree of molecularspecificity for the HP-TP or family of HP-TP where the sequence bareslittle to no homology to peptides of normal adult human proteins in theimplied probabilities of observing precise sequence alignment betweenthe intended target and off-target sequences; and 4) a predictedantigenicity comparable to or superior to known, clinically-activeT-cell antigens. The nine amino acid sequences are identified based on alinear sequence. However, it is appreciated by those skilled in the artthat the antigen is recognized based on consensus, in many cases as amotif, therefore amino acid substitutions that do not cause aconfigurational change or where a motif is intact are consideredequivalent antigens. While nine amino acids is a typical and highlyuseful length for cleaved amino acid sequences in the context of bothHLA and TCR binding, the epitope may be shorter, six, seven or eightamino acids, or part of a longer epitope, typically, ten, eleven ortwelve amino acids in length.

The sequence is linear, meaning that it is a contiguous sequence withina protein of several hundred to several thousand amino acids, really nolimit. The sequence does have conformational elements and sidechaincharge elements that allow highly specific and accurate binding to bothHLA and TCR sequences, ultimately allowing efficient binding andactivation.

Also provided is a method for reprogramming T cells to selectivelyattack tumor cells capable of perpetuating a tumor. The method includesengineering the T cells with TCR receptors that recognize the epitopesdisclosed herein.

A method for treating a cancer patient that includes reinfusing T cellsmodified to recognize the epitopes disclosed herein are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing the steps for identifying HP-Ag sequences.1, High curative potential, high potency, high probability filter; linkestablished to regeneration/perpetuation of cancer population. Curativeinput+Algorithm I; 2, Manually combine algorithm data or computationalAlgorithm II of T-cell Epitope functional Parameters (Multiple HLA ClassI types); 3, Manually computed for specificity using Basic LocalAlignment Search tool.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Highly curative (“HC”) refers to a therapy that achieves permanentregression of a cancer in a majority of patients treated.

“ACT” is used herein interchangeably to mean “Adoptive Cell Therapy” or“Adoptive Cell Transfer”, and refers to the transfer of T cells reactiveto a patient's disease state, for example, cancer back into the patient.The T cells are preferably obtained from the patient.

The term “cancer's regeneration-capable cells” (C-RC) as used hereinrefers those cells within a tumor capable of perpetuating the tumor dueto pivotal changes that misappropriate or abnormally maintain mechanismsof progenitor activation, renewal, or response.

“HP-ACT” as used herein refers to high curative potential Adoptive CellTransfer.

“HP-TP” is used herein to mean HP target protein and it refers toprotein targets expressed in a cancer, shared by individuals, that arespecific for and pivotal/essential to the perpetuation/regeneration ofthe cancer.

“HP-Ag” as used herein refers to antigens expressed within an HP-TP thathave a high probability of T cell recognition and a sequence specificitythat enables an on-target potency not limited by on- and/or off-targettoxicity.

The term “high probability” refers to a probability of eliciting a Tcell response as good or better than known positive T cell antigens.

The term “high potency” refers to an antigen that can be used clinicallyin ways that maximize its potency with little or no on- or off-targettoxicity to vital tissues.

The term “treatment” refers to the medical management of a patient withthe intent to cure, ameliorate, stabilize, or prevent one or moresymptoms of a disease, pathological condition, or disorder. This termincludes active treatment, that is, treatment directed specificallytoward the improvement of a disease, pathological condition, ordisorder, and also includes causal treatment, that is, treatmentdirected toward removal of the cause of the associated disease,pathological condition, or disorder. In addition, this term includespalliative treatment, that is, treatment designed for the relief ofsymptoms rather than the curing of the disease, pathological condition,or disorder; preventative treatment, that is, treatment directed tominimizing or partially or completely inhibiting the development of theassociated disease, pathological condition, or disorder; and supportivetreatment, that is, treatment employed to supplement another specifictherapy directed toward the improvement of the associated disease,pathological condition, or disorder.

The term “tumor” refers to an abnormal mass of tissue containingneoplastic cells. Neoplasms and tumors may be benign, premalignant, ormalignant.

The term “cancer” refers to a population of abnormal cells that displaysuncontrolled growth, invasion upon adjacent tissues, and oftenmetastasis to other locations of the body. The cancer can arise fromdifferent organs and types of tissue and can be a sarcoma, lymphoma,leukemia, carcinoma, blastoma, or germ cell tumor. The cancer can be anepithelial cancer (carcinoma) involving the parenchyma (functionaltissue) of a vital organ, such as the mammary gland of the breast, theexocrine or endocrine glands and ducts of the pancreas, hepatocytes ofthe liver, alveoli of the lung and the lining of the gut.

II. Antigens with a High Curative Potential, High Potency and HighProbability of T Cell Recognition (HP-Ag)

Peptide sequences homologous to sequences within HP target protein,having a high curative potential, high potency and high probability of Tcell recognition have been identified. These are referred to as HP-Ag,having a combination of properties that enable the design and productionof medically and commercially feasible HP-ACT. These include:

1) a core sequence of nine amino acids homologous to a sequenceexpressed within a qualified HP-TP. While the exercise can be done forall length variants, 9mer is the most common derivation of antigenic Thenine amino acid sequences are identified based on a linear sequence.However, it is appreciated by those skilled in the art that the antigenis recognized based on consensus, in many cases as a motif, thereforeamino acid substitutions that do not cause a configurational change orwhere a motif is intact are considered equivalent antigens. While nineamino acids is a typical and highly useful length for cleaved amino acidsequences in the context of both HLA and TCR binding, the epitope may beshorter, six, seven or eight amino acids, or part of a longer epitope,typically, ten, eleven or twelve amino acids in length.

The method identifies the core but the antigen binding characteristiccan be tweaked with the addition of additional sequence (usually oneamino acid to the end where the peptide binds the MEW).

2) a calculated high probability of T cell recognition and responsedetermined using calculated values for predicted peptide chemistry,probability of effective HLA presentation including: HLA bindingaffinity, processing and transport efficiency, as well as bindingstability and TCR antigenicity. Multiple values are calculated for keyvariables such as affinity and stability using available algorithms thatemploy different methods and datasets. The variables are weighted andthe level of corroboration across parameters is determined based on datafrom known positive and negative T cell antigens.

3) a high degree of molecular specificity for the HP-TP or family ofHP-TP and little to no homology to peptides of normal adult humanproteins calculated as the fold difference between specific target andnon-targets. Accordingly, the peptide sequences have a high probabilityof distinguishing normal cells from cancer cells.

HP-TP

A protein which qualifies as a HP-TP has at least the followingcharacteristics:

1) its expression is linked to a function and/or pathway necessary for atype or stage of cancer to regenerate/perpetuate itself;

2) it is expressed in a population or subpopulation of one or more typesof cancer; and

3) It is selectively expressed in such a way as to enable completekilling of expressing cells within the cancer while avoiding normalcells of vital organs.

The peptides are synthesized from in silica-qualified HP-Ag sequencesand used as tools for HP-ACT design and development.

Potential targets for T cell therapy development, including HP-TP, cancome from several sources such as viral epitopes, neoantigens caused bymutation or chromosomal rearrangements, re-expressed developmentalproteins, proteins from immune-privileged tissues such as the testis,differentiation antigens limited to non-vital cells or tissues, fusionregions of hybrid proteins, in particular, shared regions in a fusionprotein family, or epigenetically neo-expressed or re-expressed proteinssuch as cancer testis antigens (CTA) linked to enabling function.

CTA are recognized as promising targets for cancer immunotherapy becausetheir normal expression is either strictly confined to, or selectivelyexpressed in, the testis (Hoffman et al. Proc. Natl. Acad. Sci.105(51):20422-20427 (2008)). However, CTA, which are normally associatedwith spermatogenesis in development and/or the adult, cover a broadrange of proteins that differ in their function. These differingfunctions, if any, within the cancer will impact its curative potential.Some are discounted as HP-TP based on specificity and others, althoughhigh in specificity, will be discounted because of their lack offunctional connection to cancer regeneration. Expression of a CTA in acancer because of a change in methylation status for example isinsufficient alone to qualify it as an HP-TP and source for HP-Ag of themethods disclosed herein. However, some CTAs do have an establishedfunctional connection to drivers of tumor regeneration, either as animportant upstream component, or as an integral part of the growthcascade. In addition, certain cancer proteins, including some CTAs, willbe linked indirectly to regeneration and be clinically important due toan enabling auxiliary function. For example, auxiliary target proteins(Aux-TP) may support progression of the cancer by enabling the tumorcells to metastasize. However, in all cases, both HP-TP and Aux-TP mustmeet requirements for expression and specificity in step one their onlydifference being either a direct (HP-TP) or indirect (Aux-TP) role in acancer's regeneration.

Auxiliary Target Protein (Aux-TP)

The second or auxiliary target protein (aux-TP) has the mostcharacteristics of an HP-TP including frequency and specificity. Afunctional connection to cancer regeneration and progression is enablingbut not directly causative. Characteristics of Aux-TP peptides include:

-   -   1) A peptide comprising or containing a core peptide sequence of        9 amino acids homologous to a sequence expressed within a        qualified Aux-TP, where the Aux-TP is:        -   a. linked to a function and/or pathway that supports the            growth, metastasis or survival of tumor cells.        -   b. Expressed in a population or subpopulation of one or more            types of cancer,        -   c. Selectively expressed in such a way as to enable complete            killing of expressing cells within the cancer while avoiding            normal cells of vital organs    -   2) Peptide sequences with a calculated high probability of T        cell recognition and response determined using an integrated        comprehensive algorithm or a curated combination of algorithms;        and    -   3) Peptide sequences that have a high degree of molecular        specificity for the Aux-TP or a family of Aux-TP and little to        no homology to peptides of normal adult human proteins        calculated as a fold difference between the specific target and        non-targets.

III. Method of Identifying HP-Ag

Tumor-related T cell epitopes have been identified by screening tumorproteins, cDNA library cloning methods, and use of an algorithm alone orin combination to predict reactive sequences within eitherdifferentially expressed cancer proteins or neoantigens caused by avirus, mutation or translocation. Many studies have focused on thedevelopment of better diagnostics and cancer vaccines thus the need formolecular specificity or a functional connection to cancer regeneration,two requirements for an HP-Ag, have not been considered.

It is recognized that, in theory, potential targets for T cell therapydevelopment, including HP-TP, can come from several sources such asviral epitopes, neoantigens caused by mutation or chromosomalrearrangements, re-expressed developmental proteins, proteins fromimmune-privileged tissues such as the testis, and differentiationantigens limited to non-vital cells or tissues. However, in spite ofthis general awareness, efforts have yielded few if any T-cell antigens,identified or proposed, as potential T-cell antigens for immunotherapy.Where enough information is presently available to evaluate them aspotential HP-TP, most fail HP-TP criteria in a standardized assessmentof HP-TP value using Algorithm I.

Methods of T-cell target identification employed to date have failed todiscover T-cell targets and epitopes that can meet the criteriadisclosed herein and thus be useful in the development of HP-ACT orother next-generation immunotherapies. This is likely due to 1) afailure to first identify targets linked to cancer regeneration, i.e.,HP-TP, and 2) the methods used to distinguish and value potential T cellepitopes.

Ultimately, the selection of HP-TP and the subsequent isolation of HP-Agsequences capable of delivering effective, specific, and sustainedinteractions between engineered T cells and the C-RC requires amulti-faceted screening mechanism with the deliberate intent of enablinghigh curative potential. The screening acts as both a discovery tool andeffective screening mechanism in a staged procession ejecting candidateswith characteristics inconsistent with HP-TP, high probability of T-cellresponse and low on-target or off-target side effects (FIG. 1). Itallows the systematic and rapid exclusion of large amounts of data torapidly identify HP-TP as information becomes available. Morespecifically, identification of an HP-TP involves the valuation of threemajor parameters: Frequency and pattern of expression in types ofcancer, the Specificity of the Protein expression compared to normaltissues and the Functional connection of the protein's function orinvolvement in a pathway that allows one to determine whether theprotein is pivotal to the regenerative function and survival of thecancer. A positive or negative value for each major parameter is the sumof multiple characteristics that are numerically weighted based on howmuch the characteristic adds positive value or negative value to theprotein functioning as an HP-TP.

More specifically, Frequency values are calculated based on whether theTP is expressed in multiple cancers, a specific type of cancer of singleorigin, or a shared phenotype arising from multiple origins. Then the TPis graded within the category based on the frequency of expression andthe number of advanced diagnoses for the cancer target(s). The frequencycalculation determines how feasible the TP will be to test clinicallyand pursue commercially based on: number of patients, how easy it willbe to identify those patients, cost of clinical studies, and the abilityto leverage a target across multiple cancers. For example, TP that arehighly individualized with only a very small percentage of patientsexpressing the protein (for example less than 10 percent) within a largepatient population that cannot be further defined based on othercharacteristics, will have zero or negative frequency value because theTP will be impractical to screen for, test clinically and thereforeultimately treat. Commercial feasibility is a practical need required toachieve production and delivery of the therapy to a widespread patientpopulation. A TP must have a positive frequency score to proceed to Step2.

Specificity is valued based on normal expression, the novelty of adultexpression based on its being a neoantigen due to mutation orrearrangement, a re-expressed developmental protein, or a protein withnovel adult expression, such as certain cancer testis antigens normallyconfined to the testis. Normal expression and the extent of thisexpression will contribute negative values whereas a neoantigen causedby a chromosomal rearrangement expressed only in cancer will contributea positive value. The overall specificity score of the TP must bepositive to continue to Step 2.

Functional Connectivity is valued based on the degree of scientificevidence that is available to connect the protein's function to afunction pivotal to the perpetuation of the cancer, where without itsexpression, the cancer cell is unlikely to have regenerative capacity.Science that specifically demonstrates that the protein is involved indevelopmental processes or other stem cell biology adds positive value.TP involved in pathways that are enabling (like assisting migration) butnot pivotal to the survival and perpetuation of the cancer, are notassigned any positive value for this parameter. A TP must have apositive functional connectivity value to qualify as an HP-TP. However,TP determined to be involved in a non-pivotal, i.e., auxiliary functioncan proceed to Step 2 as an Aux-TP if the TP has positive Frequency andSpecificity values. Candidate TP that have insufficient scientificinformation to score its functional connectivity are put on holdawaiting additional information.

Alternatively, the selection may be done without numerical weighting ofcharacteristics by constructing a scientific argument and conclusion bycombining curated literature searches and data mining. Sequence analysisto identify HP-Ag is determined based on calculated values for predictedpeptide chemistry, probability of effective HLA presentation including:HLA binding affinity, processing and transport efficiency, as well asbinding stability and TCR antigenicity. Multiple values are calculatedfor key variables such as affinity and stability using availablealgorithms that employ different methods and datasets derived from acombination of broadly available algorithms at BIMAS (Bioinformatics andMolecular Analysis Section, NIH), SYFPEITHI, and/or Net MHC pathway(described in Tenzer, et al., Cell Mol. Life Sci. 62(9):1025-1037(2005)) among others where multiple parameters are valued. Theparameters are weighted and the level of corroboration across parametersis determined based on data from known positive and negative T cellantigens).

An exemplary method for identifying HP-TP and related HP-Ag is diagramedin FIG. 1. In general, the method includes three steps: identifyingtarget proteins as HP-TP; performing an epitope evaluation; andscreening of the HP-Ag specificity and off-target potential.

A. Step 1: Identifying HP-TPs

This first step utilizes a combination of known potential target datafrom basic and clinical research as well as specific proteomic datagenerated from specialized culture, manipulation and proteomic analysisof tumor-derived C-RC. HP-TPs are identified through (i) focused,curated literature and database searches as well as (ii) primaryexperimental data using C-RC stimulated to grow in vitro from humantumor samples. This primary data may include the derivation ofsubtractive proteomic profiles of CR-C against the tumor bulk as well asnormal tissues and experimentally-derived normal regenerative cells.Candidate proteins are further selected from the differentiallyexpressed proteins identified through literature data and/or laboratorydata.

In one embodiment, a protein is identified for its target potentialbased on (i) the parameters that determine whether the target isreachable and practical based on pattern of expression within a type ofcancer or across multiple types of cancer, the clinical ability toreasonably identify/screen for the patient population for therapy andclinically test for efficacy, (ii) its ability to discriminate cancercells from normal cells (Specificity), and (iii) the strength of itsfunctional relationship to the cancer's ability to perpetuate itself(Functional Connectivity).

Assessment at step one provides some practical assurance that thetherapy developed based on the HP-TP will have adequate commercial valueand thus be able to ultimately reach the patients that need the therapy.The method also evaluates antigen type and expression patterning as arelated but separate category, further refining the analysis andselection of high value targets. Preferably, the information associatedwith the potential target proteins is screened using the method of Step1 that assigns positive and negative numerical values to themulti-variate set of weighted parameters, either adding to orsubtracting from the curative value of the HP-TP. To qualify as anHP-TP, IS the TP must have a positive frequency value, positivespecificity, and positive confirmed or probable functional connectivitybased on known science and/or laboratory data. To qualify as an Aux-TP,the TP must have a positive frequency value, and positive specificitybut does not require positive functional connectivity.

This screen establishes the protein target as either an HP-TP or Aux-TPand assigns a target value of the candidates in the initial pool forfurther prioritization.

(i) Frequency

The TP frequency parameters include its frequency of expression, as wellas a measure of its clinical and commercial. Data is screened forexpression profiles consistent with a shared expression amongst adefinable group of patients. Measure of commercial feasibility and valueis an additional outcome and benefit of this step. In some embodiments,a protein's frequency within a cancer type and/or high expression inmultiple cancers is sufficient to positively value the protein targetfrequency. A frequency range of expression with a definable populationof ≦10% receives a negative score. A definable population refers to apatient population that can be defined based on characteristics of theirhistory and/or tumor, for example, a non-small cell lung cancer patientpopulation of never smokers that lack an Epidermal Growth FactorReceptor mutation. Positive scores are assigned based on four additionalfrequency ranges. Higher frequencies within a population have a highervalue. Frequency also values the total advanced diagnoses of thecancer(s) per year. The more advanced diagnoses, the higher the addedvalue. When the protein is expressed in more than one type of cancer,the % expression and number of advanced diagnoses are additive. Themaximum score is achieved for any target expressed in >60% of thedefinable population with total advanced diagnoses of >100,000/yr. Anegative score of <10% expression within a definable population combinedwith total advanced diagnoses of <10,000/yr worldwide will yield a zerofrequency value. Since HP-ACT is likely to be a curative therapy, evenlow scores have positive value. It is anticipated that as the experiencein HP-ACT develops and methods of screening improve, reaching patientswith rarer mutations will become increasingly feasible thereforealthough a high frequency value is more practical and allowsprioritization during the critical period of HP-ACT therapeuticdevelopment at this point in time, less frequent abnormalities might befeasibly reached in the future.

(ii) Specificity

Data is screened to determine the specificity of the target protein andin come embodiments additionally, expression profiles consistent withpotential efficacy. In one embodiment, expression of the protein iscompared between normal cells, non-cancerous but diseased cells (i.e.,cells from other disease states), and cancerous cells. Expression sharedwith normal and non-cancerous diseased cells severely limits thefeasible potency of the ACT using an antigen from the target protein,due to increased risk of collateral damage. The nature of HP-ACT therapyrequires a very stringent specificity to avoid serious collateral damageto normal tissue. To pass specificity, expression of the candidate HP-TPmust be limited to abnormal cells, normal tissues that non-vital or aresufficiently immune-privileged able to be managed to protect them from Tcell activity. The following are examples. A low level of expression innormal tissue disqualifies the TP even though the expression may be muchhigher in the cancer. Ideally, the TP is only expressed in the abnormalcancer cells of the adult or postnatal child. However a proteinexpressed in a cancer and also in the normal testis would still qualifybecause the testis is both non-vital and immune-privileged. A proteinexpressed in the cancer, the testis and the rods of the retina wouldqualify because the retina also has some degree of immune privilege andthe eye can be protected through local delivery of immunosuppressivedrugs, without risk to the rest of the body. A protein that is expressedin cancer, the testis and the glial cells of the brain would not qualifybecause of the possibility of serious injury to the brain.

(iii) Functional Connectivity

Data is screened for specific involvement in pathways or mechanismsenabling perpetuation of the tumor. A driver mutation will give a cancera growth advantage over other tumor cells. Within this group, there willbe driver mutations that are essential and ones that are non-essentialbut beneficial to tumor growth and maintenance like some epigeneticchanges caused by the primary mutation. Functional connectivity requiresthat the protein be an essential or pivotal change, capable of directlyor indirectly maintaining survival and growth capacity of thecells—where conversely, lack of expression will end the cancer cell'sgrowth and regenerative capacity. Ideally, the change is associated withthe progenitor phenotype through the prolongation or promotion of anundifferentiated state or block of differentiation through perturbationof genes associated with regeneration and differentiation such as Myc,Wnt, βCatenin, Notch, Sox2, Hedgehog, p21 etc. For example, achromosomal rearrangement that causes constitutive expression ofanaplastic lymphoma kinase (ALK) results in abnormal tyrosine kinaseactivity abnormally affecting several major signaling pathways involvedin cell cycle progression, differentiation, and survival including Ras,PLCgamma, and JNK among others (reviewed by Chiarle et al. NatureReviews Cancer 8:11-23 (2008)), normally controlled by other kinases andfeatures consistent with a regeneration-capable phenotype. ALK signalingalone can cause transformation further supporting its pivotal nature(Chiarle et al. Nature Reviews Cancer 8:11-23 (2008)). A second exampleis a translocation that causes constitutive activation of a BETbromodomain. BET bromodomains are regulatory factors for c-Myc (Delmoreet al. Cell 146:904-917 (2011)). MYC has been called the masterregulator of cell proliferation and is involved in coordinatedupregulation of many features important for regenerative capability:cell division, metabolic adaptation, and survival Delmore et al. Cell146:904-917 (2011). Therefore, an abnormally active BET bromodomain willdrive regenerative capability through MYC. Targeting the translocatedbromodomain will therefore target the regeneration-capable cells becauseof its functional connectivity to MYC. A third example is the novelexpression of an upstream regulatory protein such as an AKAP that nowcauses disregulation of a pivotal kinase, protein kinase A (PKA). PKAsbalance growth and differentiation through differential cAMP signaling(Neary et al. Oncogene 23:8847-8856 (2004)). This differential effect isalso seen in cancers (reviewed by Caretta et al. Cancers 3:913-926(2011)). Therefore abnormal neoexpression of AKAP4 (A-kinase anchoringprotein 4), a protein capable of binding and directing PKAs and normallyonly expressed in the testis, has the potential to disrupt the PKAbalance and thus the balance of growth and differentiation, an essentialaspect of organogenesis, regeneration and thus tumor formation. Aprotein capable of disrupting PKA towards an inhibition ofdifferentiation will have a functional connectivity to a cell'sregenerative capability. In these three examples, each is a proteinpivotal to the perpetuation of the cancer although through differentmeans. However in each case, this connection gives the TP a functionalconnectivity to the regeneration-capable cells of the cancer. Cells notexpressing these proteins are unlikely to be regeneration capable. Aprotein may also establish functional connectivity through other knownassociations with development, embryonic stem cell renewal orpluripotency.

B. Step 2: Epitope Evaluation

In this step, target proteins are broken down into overlappingimmunogenic peptides to ascertain the breadth of the potential T celldriven immune response. Relevant peptide characteristics evaluated inthis step include immunogenicity, chemistry and antigen processing,biochemical binding properties, and the specificity of peptide sequencein terms of potential immune response cross-reactivity. Understandingthe full spectrum of peptidic antigen characteristics enables selectionof the highest value epitopes taking into consideration how the targetprotein is recognized at the molecular level by the immune system andhow its epitopes are processed, presented, and responded to by effectorT cells to obtain true HP TCR epitopes. HP-Ag represent the activeoutput of this multifaceted screening mechanism and are the substantivephysical tool used to isolate high quality reactive TCR in the contextof various HLA (human leukocyte antigen) types. This serves as the basisfor ACT to treat intractable solid tumors specifically and effectively.

C. Step 3. Screen of HP-Ag Specificity and Off-Target Potential

The selected peptide sequences are then screened for peptide specificityand off target reactivity potential using a BLASTp screen employing theHomo sapiens RefSeq protein database and parameters optimized for shortsequence analysis and preference for minimal substitution, compositionaladjustments, and residue substitution as specificity for the intendedtarget sequence is of utmost importance. Probability values returned forboth On-target and Off-target returned results are analyzed and then acomposite value is generated reflecting the fold difference between theaverage On-target and average Off-target BLASTp generated values. Thisfold difference value can be considered the overall specificity rating.The greater the specificity rating the more specific the targetsequence. This specificity rating can also be defined as follows. Thecandidate HP-Ag sequences that passed with high specificity and lowoff-target potential were qualified as HP-Ag. A specificity rating basedon a fold difference value greater than 500 gives reasonable impliedprobability that reactivity against a protein other than the intendedtarget would be unlikely to occur. This evaluative result would then beconfirmed in further preclinical studies.

IV. Method of Using HP-Ag

The HP-Ag disclosed herein can be used as in vitro tools to enable thedevelopment of cancer immunotherapies targeting cancer regeneration. Themethods disclosed herein avoid deficiencies experienced using othermethods of epitope identification. An HP target protein (HP-TP) isestablished and its associated HP-Ag sequences are identifiedbeforehand, then TIL as well as donor PBMC (peripheral blood mononuclearcells) serve as a source of reactive T cells for T cell receptor (TCR)isolation and cloning for HP-ACT development.

Development of HP-ACT against solid tumors involves:

1. The identification of high curative potential tumor protein targets(HP-TP) that are integral/pivotal to the ability of that cancer toregenerate, i.e., perpetuate itself.

2. The identification of peptide sequences within the protein sequenceof an HP-TP that have a high probability of eliciting T cell killing(HP-Ag sequence).

3. Qualification of the sequence specificity based on the folddifference between the specific target and non-targets.

One benefit to directing ACT to peptide sequences associated with cancerregeneration is that HP-TP are more apt to be common drivers in aregenerative cancer phenotype and thus shared by individuals with acertain type of cancer and, in some cases, even across multiple types ofcancer.

HP-Ag peptides can be used singly or in combination in a variety ofmethods known to those skilled in the art to select and expand nativecytotoxic T lymphocytes (CTLs) that respond to HP-TP (HP-CTL) frompatients and donors, or alternatively, to select and clone native TCRs,for the design of TCR vectors and the engineering of HP-CTLs for use inHP-ACT. Significant value can be placed on the ability to isolateantigen targets that lead directly to high value TCRs reactive to thosetargets, however to do so against multiple expressed targets furtherincreases the chance of curative results. Combining intracellular aswell as surface expressed antigen targets can be used to optimize andspecifically tailor the treatment to the specific cancer sub-type andstage and minimize disease relapse and/or metastasis.

In a preferred embodiment, the HP-Ag sequences are used as tools toselect naturally occurring TCRs for the subsequent design and productionof modified or unmodified CTLs for adoptive cell transfer. One or moreHP-Ag peptides can be used alone or incorporated into molecular andcellular technologies and systems to selectively expand and adoptivelytransfer back to the patient large numbers of CTLs that respond topresented HP-Ag epitopes or set of HP-Ag epitopes. HP-Ag peptides canalso be incorporated into peptimers or loaded into antigen presentingcells and cell lines to isolate and clone T cell receptors (TCRs). ThecDNA from the cloned receptors can then be incorporated into vectors togenetically engineer patient T cells that will now recognize and killtumor cells expressing the HP-TP. Current vector technologies utilizinglentiviral expression and packaging systems allow for a wide variety ofselective and targeted protein expression combinations controlled byseparate promoter sequences. This can now be done in such a way thatmulti-chain proteins such as TCRs along with secondary augmenting oradjuvant proteins can be expressed from a single vector under theguidance of separate control elements allowing optimization of TCRexpression. The latter case does not require the patient to have nativeT cells that respond to the HP-TP of their cancer.

Examples of how the disclosed epitopes may be used in T-cell focusedimmunotherapies include the use of HP-Ag for selection TCRs for thesubsequent development of non-cell-based soluble TCR technologies suchas ImmTAC (Immune mobilizing monoclonal TCR (T cell receptors) Againstcancer) (Immunocore) or the use of surface-expressed HP-TPs as antigensto design ACT therapies based on the use of chimeric antigen receptors(CAR-ACT) (Reviewed in Shi, et al., Molecular Cancer, 13:219 (2014)—boththerapies acting at the T cell level. Preferably, the HP-Ag are used inHP-ACT therapies employing cloned native TCRs alone or in combinationwith co-expressed immunomodulatory cytokines.

The immune system includes two key recognition systems, antibodies,which target cell surface proteins, and T cell receptors, which targetHLA-presented peptide antigens potentially derived from virtually anyintracellular protein. ImmTACs are HLA-peptide targeting bi-specificbiologics which include an engineered T cell receptor based targetingsystem fused to an anti-CD3 scFv based effector function. ImmTACsfunction by binding to defined HLA-peptides with extremely high affinity(typically <50 pM), simultaneously decorating the target cell with loweraffinity (nM) CD3 specific scFv fragments. Any T cell that comes intodirect physical contact with an ImmTAC-decorated cancer cell isautomatically redirected to kill the cell, regardless of the T cell'snative antigen specificity.

In some cases it is desirable to direct T cell killing to more than onetarget. At a minimum, one target must be an HP-TP for it to be an HP-ACTtherapy. However, it may be desirable to eliminate the entire cancer(all cancerous cells of the tumor) using ACT. While the expansion oftumor T cell killing to other targets, a phenomenon known as antigenspreading, is likely during HP-ACT, it may be desirable to ensure moredirected T cell killing to stop metastasis, better ensure theelimination of the bulk of the tumor or rapidly attenuate bulk tumorgrowth to eliminate the possibility of future changes or mutations inthe remaining cells that could render them regeneration-capable. Thiscan be achieved by the inclusion of T cells that respond to an enablingauxiliary function.

It will be evident to those skilled in the art that the use of the HP-TPand/or HP-Ag as described in the present invention need not be limitedto HP-ACT and can be used to improve the clinical potential of manytypes of cancer immunotherapy through improved targeting of a specific Tcell response to cancer regeneration.

EXAMPLES Example 1 Distinguishing High Curative Potential TargetProteins (HP-TP) and Aux-TP from Non-HP-TP and Non-Aux-TP UsingMesothelin as the Example

The cell surface protein mesothelin has been identified and developed asa target for ACT. Mesothelin is used to illustrate the differencebetween simply a “cancer marker” or TP and an HP-TP or Aux-TP and howthey are qualified. The process applied in this example is not limitedto the protein of the example but is generally applicable to allexpressed cancer proteins.

Mesothelin is a cell surface protein highly expressed in mesothelioma,as well as ovarian, pancreatic, and a subset of lung cancers (Somers etal. Biomarker Insights 9:29-37 (2014)). It is a cell surface proteinthat begins as a precursor that is then split into thecell-membrane-associated protein mesothelin and a soluble megakaryocytepotentiation factor (Somers et al. Biomarker Insights 9:29-37 (2014)).Experts in the field of cancer immunotherapy consider surface-boundmesothelin a clinically viable candidate for ACT, particularly employingchimeric antigen receptor (CAR) modified T cells because of its surfaceexpression (CAR-ACT requires surface expression of the TP because of itsreliance on antibody-based target recognition for the initiation of Tcell killing.) The supposition is that mesothelin is targetable by ACTbecause it is highly expressed in cancer compared to normal mesothelium.However, there are several aspects of mesothelin as a TP for ACT thatcould discount its value as either a HP-TP or Aux-TP. Testing of thetarget protein is a necessary first step in determining whether theidentification of HP-Ags for HP-ACT development is possible andfeasible.

Mesothelin's target potential was analyzed based on the parameters offrequency, pattern of expression, and its clinical and commercialfeasibility (Frequency), its ability to discriminate cancer cells fromnormal cells (Specificity), and the strength of its functionalrelationship to the cancer's ability to perpetuate itself (FunctionalConnectivity). To qualify, the TP must have a positive frequency basedon the degree the target is shared within a cancer population and thesize of the population, specificity, and a high confirmed or probablefunctional connectivity.

Step 1. Qualification of Mesothelin as an HP-TP or Aux-TP

A. TP Frequency

Mesothelin expression in cancer qualifies it as a potential TP based onfrequency of expression in multiple cancers. Mesothelin is a proteolyticcleavage product of a mesothelin precursor which when cleaved gives riseto a secreted megakaryocyte potentiation factor and the GPI-membraneanchored mesothelin, the potential cancer protein target. Mesothelin iselevated in mesothelioma and is currently used in its diagnosis,prognosis and monitoring (Hollevoet et al. Am. Respir. Crit. Care Med.181:620-625 (2010); Creaney et al. Clin. Cancer Res. 17:1181-1189(2011)). It is also highly expressed in ovarian cancer (Chang et al.Proc. Natl. Acad. Sci. USA 93:136-140 (1996)), pancreatic cancer (Arganiet al. Clin. Cancer Res. 7:3862-3868 (2001)) and the majority of lungadenocarcinomas (Ho et al. Clin. Cancer Res. 13:1571-1575 (2007)). Itsfrequency within a cancer type and high expression in multiple cancersis sufficient to positively value mesothelin target frequency for ACT.

B. TP Specificity

Mesothelin is expressed at lower levels in normal mesothelium of theperitoneum, pericardium and pleura and possibly the trachea (Chang etal. Proc. Natl. Acad. Sci. USA 93:136-140 (1996)). Also, its expressionis shown to increase in renal disease (Somers et al. Biomarker Insights9:29-37 (2014)). Expression shared with normal and non-cancerousdiseased cells severely limits the feasible potency of the ACT due torisk of collateral damage to the peritoneal lining, pleura andpericardium as well as the kidney. This is particularly important in thecancer treatment as many chemotherapeutics, which the patients may havebeen treated with prior to ACT therapy are known nephrotoxins, where thecompromised kidney will also express elevated levels of mesothelin.Differential expression is not enough to overcome the reduction in valuebecause of a loss of both potential potency and potential on-targetcollateral damage due to lack of specificity. Importantly, the increasedexpression in the impaired kidney indicates that mesothelin upregulationmay be a more generalized wound-healing-associated response and mostlikely not limited to just the impaired kidney. This lack of specificitygives mesothelin a strong negative value as a TP for ACT.

C. TP Functional Connectivity

Mesothelin failed specificity alone would be sufficient to disqualify itas either an HP-TP and Aux-TP, however, the analysis of its functionalconnectivity was performed for purposes of the example. Mesothelin'sfunctional connectivity was measured based on its relationship andsignificance to normal function, tumor function, and in particular,cancer regeneration. Sufficient information existed to assess itsprobable connection to cancer regeneration and determine its functionalconnectivity through analysis of protein function, connection to keydevelopmental (regenerative), cell proliferation and survival pathways.A curated literature search found that mesothelin is functionally linkedto aspects of tissue remodeling associated with a wound healing responsethrough its association with elevated levels of MMP 7 and IL6-IL6R.Upregulation of a single MMP is not likely to be an essential driverintegral to a cancer's ability to regenerate. Even if expressed inmetastatic C-RC, mesothelin's biological role in MMP-7 upregulation isless likely to be constant within the C-RC population of the tumor,particularly if they are not actively undergoing metastasis. Thereforethis functional connection added no positive value to mesothelin as anHP-TP target.

An increase in mesothelin expression correlates with a rise in IL6-IL6Rexpression and its actions through the activity of NFkappaB, a majorsignaling hub in the wound healing response. This response is notspecific to cancer as evidenced by the rise in mesothelin as well as IL6(Ranganathan et al. Am. J Physiol. Renal Physiol. 304:F1054-F1065(2013)) in kidney disease and its constitutive baseline expression inmesothelial linings. Mesothelin expression leading to IL6 expression andaction is a wound healing phenotype that enables cell attachment,survival and continued growth in an inflammatory environment. Knockoutstudies in mice have found no observed effect on growth and development.Therefore mesothelin upregulation is likely in response to a pivotalchange that will drive the cancer rather than the cause of it. Eventhough it can lead to an increase in IL6, the cytokine levels can beincreased for other reasons. This eliminates its values as an HP-TP anddiscounts mesothelin's value as a necessary auxiliary function in thecancer.

Mesothelin is reported to bind MUC16 (CA125) (Gubbels et al. MolecularCancer 5:50-64). CA125 is described as an ovarian cancer tumor marker.Mesothelin binding to MUC16 is believed to contribute to the cell-celladherence of metastatic cells to increase metastatic tumor mass as wellas the adherence of ovarian cancer cells to the peritoneum. (Felder etal. Molecular Cancer 13:129-143 (2014)). However MUC16 is expressed innormal endometrium, lung and amnion and mesothelia among other tissues(Wang et al. Differentiation 76(10):108101092 (2008)). The interactionbetween mesothelin and MUC16 observed in ovarian cancer is therefore anupregulated normal function, devaluing it as an Aux-TP capable ofdiscriminating the C-RC of a cancer. Differential expression is notsufficient to positively value the target protein.

When all factors are valued for their positive and negative measures offrequency, specificity and functional connectivity, mesothelin passesthe frequency measure, fails to qualify based on specificity, and failsfunctional connectivity. Mesothelin would not move forward to evaluationof the protein sequence for high probability HP-Ag sequences (Step 2).This is in sharp contrast to the justification and pursuit of mesothelinas a viable ACT target by several groups. Rather, Step 1 predicts thatthe mesothelin target will be incapable of generating an HP-ACT therapy.

Example 2 Comparison of HP-Ag Derivation Against an Alternative Methodof Target and Epitope Identification for ACT Targeting Cancer Stem Cells

Many methods to date have had the intent of improving cancer vaccinesrather than ACT therapy so their deficiencies in discrimination of HP-TPand HP-Ag are not surprising. However, some approaches have beendesigned with the goal of identifying cancer proteins and epitopes forACT targeting cancer stem cells. One such example is the work ofOchsenreuther et al. (2008) (Ochsenreither, et al. Blood119(23):5492-5501 (2012)) where they describe a protein and epitopediscovery approach for ACT therapy to target leukemic stem cells inacute myeloid leukemia. Both the target and HLA A2 9 amino acid (9mer)epitopes identified by Ochsenreuther et al. Blood 119(23):5492-5501(2012) were compared using the stepwise, gated approach and associatedanalysis disclosed herein. The complete protein sequence was thenanalyzed using Step 2 of the methods herein to determine whether thisapproach would have identified similar or different antigenic sequences.The results illustrate the impact of the approach on both practical andscientific terms, the difference in resulting output, as well as thebenefits and efficiency of the disclosed methods to identify HP-Ag.

Ochsenreuther et al. (2012) employed microarray expression analysisincluding more than 100 probe sets of leukemic stem cells, hematopoieticstem cell subpopulations, and peripheral tissues to ultimately identifya single candidate, Cyclin A-1 (CCNA1), the only target found aftersubsequent RT-PCR. Cyclin A-1 is detected in over 50% of AML patients,is associated with cell proliferation, produces leukemia in mice and isminimally expressed in normal tissues other than the testis. Thisassessment of the TP led Ochsenreuther et al. (2012) to characterize itas a cancer-testis antigen and more specifically, a leukemia-testisantigen suitable for ACT development. They then pulsed dendritic cellswith Cyclin A-1 peptides and used the pulsed cells to stimulate clonesof reactive T cells from two normal donors. The method identified 8immunogenic peptides across at least 3 HLA types. Focusing on HLAA*0201, they noted that their cell-based selection method was able toidentify a reactive 11 amino acid sequence (11mer) that was notpredicted in their use of three in silico methods (SYFPEITHI, BTMAS,IEDB analysis resource) although the in silico methods did identify a10mer and 15mer at this location.

For comparison, Cyclin. A-1 and its epitopes were screened according tothe methods disclosed herein. Cyclin A-1 was first evaluated as an HP-TPbased on the parameters of frequency, pattern of expression, and itsclinical and commercial feasibility (Frequency), its ability todiscriminate cancer cells from normal cells (Specificity), and thestrength of its functional relationship to the cancer's ability toperpetuate itself (Functional Connectivity).

Step 1. Qualification of Cyclin A-1 as an HP-TP or Aux-TP

A. Frequency

Expression of the protein in 50% of AML was sufficient to qualify it forfrequency. Its expression has also been described in other cancers suchas prostate (Weigiel et al. JNCI 100(14):1022-1036 (2008)), breast(Khaja et al. PLoS ONE 8(8):e72210 (2013)) and non-small cell lungcancer (Kosacka et al. in vivo 23:519-526 (2009)), which added to itspositive frequency.

B. Specificity

Cyclin A-1's presumed specificity was noted by Ochsenreuther et al.(2012) as a compelling characteristic for targeted ACT. However, acurated mining of the literature and other available information foundevidence that Cyclin A-1 was not restricted to the normal testis. CyclinA-1 is expressed at low levels in normal human hematopoietic tissue,which is not surprising given its strong association with leukemia.While this would add to its functional connectivity, specificity isdiscounted because of it. When Cyclin A-1 was first discovered as newform of Cyclin A (Yang et al. Cancer Res. 57:913-920 (1997)). It wasreported that Cyclin A-1 mRNA was found by northern blot analysispreferentially in testis but to a lesser extent also in the normalbrain. In van der Meer et al. Reproduction 127:503-511 (2004) reportedits expression at low levels in normal mice in the olfactory bulb,hippocampus and amygdala of the adult brain. More recently, Cyclin A-1expression has been linked to circadian rhythm and sleep in Drosophila(Rogulja et al. Science 335(6076):1617-1621 (2012)). In 2001 a studylooking at the differential methylation status of the Cyclin A-1promoter reported that although Cyclin A-1 was predominantly expressedin the testis, modest levels could be detected by RT-PCR in the spleen,prostate, leukocytes, colon and thymus (Müller-Tidow FEBS Letters490:75-78 (2001)). Combined, this data suggests that while Cyclin A-1 ispreferentially expressed in the testis, it would not be unexpected tofind the protein in other normal tissues, of most concern, in portionsof the brain and hematopoietic tissue. This would discount it as anHP-Ag candidate based on inadequate specificity.

C. Functional Connectivity

Cyclin A-1 is associated with meiosis in sperm and linked toregeneration. For example, its expression appears needed for inducedpluripotent stem cells to achieve a non-tumorigenic pluripotent state(MeLenachan Stem Cells and Development 21(15):2891-2899 (2012)) andCyclin A-1 is expressed in normal CD34+ hematopoietic stem cells (Yanget al. Blood 93:2067-2074 (1999)) that establish a connection toregeneration, at least in the hematopoietic system. It other tissuesCyclin A-1 appears to have different functions that would not beconnected to mechanisms of regeneration. There is sufficient knowledgeto connect Cyclin A-1 to the C-RC in the case of leukemias.

Cyclin A-1 meets the criteria of an HP-TP in frequency and functionalconnectivity (when restricted to leukemia). However Cyclin A-1 hasinsufficient specificity to qualify it as either an HP-TP or Aux-TPbecause of its expression in the normal brain (with confirmation neededin humans), its potential to interfere with hematopoiesis, whichdiscounts its potential potency, and indication that it can be expressedin other tissue like the colon depending on circumstances. Thereforesuccessful use of Cyclin A-1 would require further information and studyin order to qualify it as an HP-TP with a high likelihood that it wouldnot qualify as more is known. Cyclin A-1 would not proceed to Step 2 inthe methods disclosed herein. Nevertheless, this example proceeded toStep 2 epitope discovery in order to compare the methods disclosedherein, to the methods of Ochsenreuther et al (2012) for epitopediscovery.

These studies focused on HLA A2 epitopes identified by both approaches.Ochsenreuther et al. (2012) identified 4 HLA A2 9mers: YAEEIYQYL (SEQ IDNO:1), AETLYLAVN (SEQ ID NO:2), FLDRFLSCM (SEQ ID NO:3) and ASKYEEIYP(SEQ ID NO:4) as well as one 11mer, SLIAAAAFCLA (SEQ ID NO:5). Using acomprehensive comparative analysis of multiple, corroborativeparameters, two of the four 9mers were identified as being highprobability T cell epitopes: FLDRFLSCM (SEQ ID NO:3) and sequenceYAEEIYQYL (SEQ ID NO:1) by the methods of Step 2. The remaining two9mers showed a low probability of being strong T cell epitopes based onweak calculated binding affinity, stability (dissociation half-times) aswell as predicted antigenicity and chemistry and thus would not qualifyas candidate HP-Ag using the methods disclosed herein. It also points tothe idea that in vitro selection to identify epitopes may not guaranteerobust T cell reactivity.

The use of three well-established algorithms, SYFPEITHI (Rammensee,Bachmann, Stevanovic: MHC ligands and peptide motifs. Landes Bioscience1997 (International distributor—except North America: Springer VerlagGmbH & Co. KG, Tiergartenstr. 17, D-69121 Heidelberg), BIMAS (Parker, KC., M. A. Bednarek, and J. E. Coligan. J. Immunol. 152:163 (1994.) andIEDB (Tenzer et al. Cel Mol Life Sci 62(9):1025-37 (2005)) failed toidentify the 11mer, a fact Ochsenreuther et al. (2012) used to supporttheir case for the superiority of biological fishing for theidentification T cell antigens. However, the in silico process disclosedherein not only identified a high probability core 9mer sequence withinthe 11mer peptide (SLIAAAAFCLA (SEQ ID NO:5)): LIAAAAFCL (SEQ ID NO:6),it also identified an additional high probability candidateincorporating a portion of the 11mer sequence: YLPSLIAA (SEQ ID NO:7).This illustrates that the deficiency is not in the use of in silicomethods per se but that one needs more comprehensive in silico methods,combined in a corroborative system preferably tested using positive andnegative controls.

Step 2 identified additional candidates with properties equal to orsuperior than those previously found by the investigators. In practice,when the identified core 9 mer sequences are used for selection of Tcells, that testing can include the addition of peptides on either endof the 9mer core. Therefore unlike the Ochsenreuther approach, theprocess disclosed herein has a much higher likelihood of capturing themost robust antigen(s) for T cell selection. Very few 9mers (the mostlikely to bind well to CD8+TCRs (Doan et al. Lippincott's IllustratedReviews: Immunology Second Edition Wolters Kluwer Baltimore (2013)) andin particular, A2 epitopes had been identified by the laboriousOchsenreuther process. In contrast, Step 2 of the process disclosedherein identified several additional candidate HP-Ag in HLA A2,increasing the likelihood of yielding antigenic peptides with a highprobability of TCR reactivity.

HLA A2 high probability 9mer peptides within Cyclin A-1 were selectedfrom a total of 457 sequences using Step 2. Sequences that were selectedboth manually and by Algorithm II are shown in Table 1. Ochsenreuther etal. (Ochsenreither, et al. Blood 119(23):5492-5501 (2012)) sequences areincluded in bold.

TABLE 1 9mer peptides within Cyclin A-1 with HLA specificity HP Sequencebased HLA on the Specif- Core 9mer method of Target icity sequenceStep 2 Cyclin A-1 A2 AIMYPGSFI Yes (SEQ ID NO: 8) Cyclin A-1 A2YLSWEGPGL Yes (SEQ ID NO: 9) Cyclin A-1 A2 MAFAEDVYEV Yes(SEQ ID NO: 10) Cyclin A-1 A2 TLKSDLHFL Yes (SEQ ID NO: 11) Cyclin A-1A2 SLGTDVINV Yes (SEQ ID NO: 12) Cyclin A-1 A2 YQYLREAEI Yes(SEQ ID NO: 13) Cyclin A-1 A2 RTILVDWLV Yes (SEQ ID NO: 14) Cyclin A-1A2 ILVDWLVEV Yes ((SEQ ID NO: 15) Cyclin A-1 A2 KLRAETLYL Yes(SEQ ID NO: 16) Cyclin A-1 A2 FLDRFLSCM Yes (SEQ ID NO: 3) Cyclin A-1 A2VLRGKLQLV Yes (SEQ ID NO: 17) Cyclin A-1 A2 QLLKMEHLL Yes(SEQ ID NO: 18) Cyclin A-1 A2 KVLAFDLTV Yes (SEQ ID NO: 19) Cyclin A-1A2 NLAKYVAEL Yes (SEQ ID NO: 20) Cyclin A-1 A2 SLLEADPFL Yes(SEQ ID NO: 21) Cyclin A-1 A2 YLPSLIAAA Yes (SEQ ID NO: 22) Cyclin A-1A2 LIAAAAFCL Yes (SEQ ID NO: 6) Cyclin A-1 A2 FTGYSLSEI Yes(SEQ ID NO: 23) Cyclin A-1 A2 SLSEIVPCL Yes (SEQ ID NO: 24) Cyclin A-1A2 SLMEPPAVL Yes (SEQ ID NO: 25) Cyclin A-1 A2 YAEEIYQYL Yes(SEQ ID NO: 1) Cyclin A-1 A2 AETLYLAVN No (SEQ ID NO: 2) Cyclin A-1 A2ASKYEEIYP No (SEQ ID NO: 4) *The combination estimates aspects ofepitope chemistry, biochemistry, processing, and immunogenicity.Bold indicates epitopes also identified by Ochsenreuther et al.(Ochsenreither, et al. Blood 119(23):5492-5501 (2012)) althoughLIAAAAFCL (SEQ ID NO:6) was identified within a 11 mer.

This example illustrates a key difference between the methods disclosedby Ochsenreuther and the methods disclosed herein. The Ochesenreutherapproach relies on the T cell reactivity to define the antigenictargets, leaving open the possibility for individual bias in immuneresponse, the second relies on unbiased in silico chemistry andbiochemistry, which is only then followed by a search of T cellsreacting to the specific antigen. The identification of multipleepitopes increases the likelihood of finding suitable TCRs against thetarget.

Example 3 The Derivation of HP-Ag Peptides Homologous to Sequenceswithin the Fusion Region of the BRD4-NUT Fusion Protein Expressed in NUTMidline Cancers

BRD4-NUT ((bromodomain containing 4 protein-nuclear protein in testis)is a fusion protein present in a subset of NUT midline cancers. NUTmidline carcinomas are non-operable with few treatment options (FrenchNature Reviews Cancer 14:149-150 (2014)). If BRD4-NUT were a feasible,safe and potent ACT target, it would offer a valuable treatment optionfor NUT midline carcinoma. These studies were commenced by evaluatingthe fusion protein for its target potential based on the parameters offrequency, pattern of expression, and its clinical and commercialfeasibility (Frequency), its ability to discriminate cancer cells fromnormal cells (Specificity), and the strength of its functionalrelationship to the cancer's ability to perpetuate itself (FunctionalConnectivity).

Step 1. Qualification of BRD4-NUT as an HP-TP or Aux-TP

A. TP Frequency

The BRD4-NUT fusion protein is expressed in approximately 50% of NUTmid-line carcinomas. This high frequency of expression within NUTmidline carcinomas gave it a sufficient positive frequency value. Thereported frequency of BRD4-NUT cancers is also likely to rise withincreased screening, now prompted because of the availability of cancerdrugs that target active bromodomains

B. TP Specificity

NUT is a CTA with expression confined to the testis under normalcircumstances although the significance of its expression in and ofitself is unknown. Targeting abnormally activated BRD4 expression for Tcell killing is not desirable due to the broad expression of BRD4 innormal cells and the potential for serious side effects. Targeting NUT,a cancer testis antigen, is more feasible. However expression of NUTalone does not necessarily target C-RC, the cells with the mostfunctional significance for the patient. Finding HP-Ag sequences withhomology to sequences within the unique fusion region of BRD4-NUTensures that the ACT will target cells that have the active bromodomaindriving the cancer while leaving normal BRD4 activity unrecognized,giving BRD4-NUT a positive specificity value.

C. TP Functional Connectivity

BRD4 fusion with the cancer testis antigen NUT results in abnormalbromodomain activity. The bromodomain motif is a key aspect ofepigenetic regulation. In development, lack of BRD4 is lethal. BRD4 hasbeen reported as a key regulator of embryonic stem cell (ES) renewal andpluripotency regulated principally through Nanog expression (Liu et al.Cell Death Differ. 21(12):1950-1960 (2014)). BRD4 is downregulated uponES differentiation. In cancers, BRD4 regulates c-Myc and selectivelybinds large clusters of enhancers that control tumor oncogenes (Liu etal. Cell Death Differ. 21(12):1950-1960 (2014)). Malregulated BRD4 leadsto a loss of proliferative control at least in part, through mechanismsrelated to stem cell biology. Bromodomain activity has been establishedas a cancer drug target. Yan et al. (Yan et al. J. Biol. Chem.286:27663-27675 (2011)) have described BRD4's ability to blockdifferentiation of NUT midline carcinoma cells through downstreamrepression of c-fos. Because the abnormally active bromodomain is apivotal change capable of driving the cancer, the likelihood that itwill be active in the C-RC of BRD4-NUT cancers is high. Its associationwith development and embryonic stem cell renewal provides an additionallink to C-RC biology. The epigenetic impact of the driver bromodomain ofBRD4-NUT established the fusion protein's functional connectivity toC-RC biology. This connection to the C-RC can be further corroborated inC-RC derived from Nut midline carcinoma using technology that activatesa regenerative response in vitro.

The potential therapeutic value of the BRD4-NUT was positive forfrequency, specificity and functional connectivity. Positive assessmentof Frequency, Specificity and Functional Connectivity qualified BRD4-NUTto advance to Step 2.

Step 2. Identification of Candidate HP-Ag Sequences

The BRD4-NUT fusion region sequence used to identify high probabilitycandidate HP-Ag:

(SEQ ID NO: 26) EPSLKNSNPDEIEIDFETLKPSTLRELERYVTSCLRKKRKPQAEKVDVIAGSSKMKGFSSSESESSSESSSSDSEDSETASALPGPDMSMKPSAALSPSPALPFLPPTSDPPDHPPREPPPQPIMPSVFSPDNPLMLSAFPSSLLVTGDGGPCLSGAGAGKVIVKVKTEGGSAEPSQTQNFILTQTALNSTAPGTPCGGLEGPAPPFVTASNVKTILPSKAVGVSQEGPPGLPPQPPPPVAQLVPIVPLEKAWPGPHGTTGEGGPVATLSKPSLGDRSKISK DVYENFRQWQRYKALARRHLSQSP

Overlapping 9 peptide sequences within the fusion region where evaluatedmanually and using a comprehensive integrated algorithm that assignedweighted values to the sequence's chemistry, antigen processing, HLAspecificity, and binding kinetics and that incorporated known positiveand negative T cell epitopes as controls. A total of 298 sequenceswithin the BRD4-NUT fusion region were screened.

TABLE 2  Candidate HP-Ag sequences (9-mer sequences)in the BRD4-NUT fusion region with their HLA A2 specificity HP Sequencebased on the  HLA Core 9mer  method Target Specificity sequenceof Step 2* BRD4-NUT A2 TLRELERYV Yes (SEQ ID NO: 27) BRD4-NUT A2MLSAFPSSL Yes (SEQ ID NO: 28) BRD4-NUT A2 SAFPSSLLV Yes (SEQ ID NO: 29)BRD4-NUT A2 ILPSKAVGV Yes (SEQ ID NO: 30) BRD4-NUT A2 ALPGPDMSM Yes(SEQ ID NO: 31) BRD4-NUT A2 MSMKPSAAL*** Yes (SEQ ID NO: 32) BRD4-NUT A2AALSPSPAL*** Yes (SEQ ID NO: 33) BRD4-NUT A2 AQLVPIVPL Yes(SEQ ID NO: 37) *The combination estimates aspects of epitope chemistry,biochemistry, processing, and immunogenicity. **Algorithm IIincorporates estimates of the manual curation but has the additionalbenefit of being formulated and tested against known positive andnegative T cell epitopes to remove potential bias and further improveselection accuracy. ***Identified as a candidate sequence for more thanone HLA type.

Several sequences were identified as having comparable molecularcharacteristics as good or better than well-characterized epitopes withknown in vivo immunogenicity and in particular, T cell reactivity. Uponanalyzing multiple target proteins, the data showed that not allparameters were consistent between proteins, emphasizing the need formultiple, corroborative data points. Sequences that did not reachconsensus were re-examined manually. Sequences from some target proteinsshowed a very high consensus between Algorithm II ***and manualselection whereas in others, the algorithm identified additionalsequences not selected manually. This was true of BRD4-NUT. Algorithm II***identified one sequence that was simply missed in the manualselection (AQLVPIVPL (SEQ ID NO:37). In addition, it identified 3sequences that were not selected because of border-line values in someparameters discounted in the manual selection. These sequences were nowconverted to “yes” with the support of Algorithm II*** (whichmathematically takes into account positive and negative controls). Ofinterest was the fact that two of the three conversions were identifiedmanually for other HLA types. Sequences not reaching consensus were puton hold. The sequences able to reach consensus for A2, or positivelyidentified manually in other HLA types, advanced to Step 3.

Available data for HLA-A2 are the most complete data available,including the availability of control data. This data was used toconstruct Algorithm II***. However, there were sufficient available datacovering most parameters to manually select epitopes for additional HLAtypes from the comprehensive data set. Results using the schemevalidated for A2 by Algorithm II can be used for the manual curation ofnon-A2 sequences. In turn the selections can then be used to adjustAlgorithm II*** to handle the non-available data points and accommodateevaluation of additional HLA types. Most common HLA types could beanalyzed. Further experiments focused on major HLA types that, inaddition to A2, would be present in a majority of patients in NorthAmerica and Europe (Table 3).

TABLE 3  Candidate HP-Ag sequences (9-mer sequences) in BRD4-NUTfusion region with their HLA specificity HP Sequence HLAbased on methods Target Specificity Core 9 mer sequence of Step 2*BRD4-NUT A3 CLSGAGAGK (SEQ ID NO: 38) Yes BRD4-NUT A3VIAGSSKMK (SEQ ID NO: 39) Yes BRD4-NUT A3 YVTSCLRKK (SEQ ID NO: 40) YesBRD4-NUT B7 KPQAEKVDV (SEQ ID NO: 41) Yes BRD4-NUT B7MSMKPSAAL*** (SEQ ID NO: 32) Yes BRD4-NUT B7 KPSAALSPS SEQ ID NO: 42)Yes BRD4-NUT B7 AALSPSPAL*** (SEQ ID NO: 33) Yes BRD4-NUT B7SPSPALPFL (SEQ ID NO: 43) Yes BRD4-NUT B7 SPALPFLPP (SEQ ID NO: 44) YesBRD4-NUT B7 PPQPIMPSV (SEQ ID NO: 45) Yes BRD4-NUT B7APGTPCGGL (SEQ ID NO: 46) Yes BRD4-NUT B7 GPAPPFVTA (SEQ ID NO: 47) YesBRD4-NUT B7 LPPQPPPPV (SEQ ID NO: 48) Yes BRD4-NUT B7QPPPPVAQL (SEQ ID NO: 49) Yes BRD4-NUT A3A G A G K V I V K (SEQ ID NO: 200) Yes BRD4-NUT A3N V K T I L P S K (SEQ ID NO: 201) Yes BRD4-NUT A3L V P I V P L E K (SEQ ID NO: 202) Yes BRD4-NUT A11I E I D F E T L K (SEQ ID NO: 203) Yes BRD4-NUT A11E T L K P S T L R (SEQ ID NO: 204) Yes BRD4-NUT A11R Y V T S C L R K (SEQ ID NO: 205) Yes BRD4-NUT A11Y V T S C L R K K (SEQ ID NO: 206) Yes BRD4-NUT A11T S C L R K K R K (SEQ ID NO: 207) Yes BRD4-NUT A11V I A G S S K M K (SEQ ID NO: 39) Yes BRD4-NUT A11C L S G A G A G K (SEQ ID NO: 38) Yes BRD4-NUT A11A G A G K V I V K (SEQ ID NO: 200) Yes BRD4-NUT A11N V K T I L P S K (SEQ ID NO: 201) Yes BRD4-NUT A11L V P I V P L E K (SEQ ID NO: 202) Yes BRD4-NUT A24L S P S P A L P F (SEQ ID NO: 208) Yes BRD4-NUT A24P Q P I M P S V F (SEQ ID NO: 209) Yes BRD4-NUT A24V F S P D N P L M (SEQ ID NO: 210) Yes BRD4-NUT A24F S P D N P L M L (SEQ ID NO: 211) Yes BRD4-NUT A24L S A F P S S L L (SEQ ID NO: 212) Yes BRD4-NUT A24V T A S N V K T I (SEQ ID NO: 213) Yes BRD4-NUT A24I S K D V Y E N F (SEQ ID NO: 214) Yes BRD4-NUT B7S V F S P D N P L (SEQ ID NO: 215) Yes BRD4-NUT B7M L S A F P S S L (SEQ ID NO: 28) Yes BRD4-NUT B7P P V A Q L V P I (SEQ ID NO: 216) Yes BRD4-NUT B7V A T L S K P S L (SEQ ID NO: 217) Yes BRD4-NUT B7R Q W Q R Y K A L (SEQ ID NO: 218) Yes BRD4-NUT B8L E R Y V T S C L (SEQ ID NO: 219) Yes BRD4-NUT B8C L R K K R K P Q (SEQ ID NO: 220) Yes BRD4-NUT B8L R K K R K P Q A (SEQ ID NO: 221) Yes BRD4-NUT B8R K K R K P Q A E (SEQ ID NO: 222) Yes BRD4-NUT B8M S M K P S A A L (SEQ ID NO: 32) Yes BRD4-NUT B8M L S A F P S S L (SEQ ID NO: 28) Yes BRD4-NUT B8N F I L T Q T A L (SEQ ID NO: 223) Yes BRD4-NUT B8R Q W Q R Y K A L (SEQ ID NO: 218) Yes BRD4-NUT B8A L A R R H L S Q (SEQ ID NO: 224) Yes BRD4-NUT B15A L P G P D M S M (SEQ ID NO: 31) Yes BRD4-NUT B15P Q P I M P S V F (SEQ ID NO: 209) Yes BRD4-NUT B15M L S A F P S S L (SEQ ID NO: 28) Yes BRD4-NUT B15T Q T A L N S T A (SEQ ID NO: 225) Yes BRD4-NUT B15G L E G P A P P F (SEQ ID NO: 226) Yes BRD4-NUT B15A Q L V P I V P L (SEQ ID NO: 37) Yes BRD4-NUT B15R S K I S K D V Y (SEQ ID NO: 227) Yes BRD4-NUT B15I S K D V Y E N F (SEQ ID NO: 214) Yes BRD4-NUT B15R Q W Q R Y K A L (SEQ ID NO: 218) Yes BRD4-NUT B15W Q R Y K A L A R (SEQ ID NO: 228) Yes NA = not yet available.***Identified as a candidate sequence for more than one HLA type.

Step 3. Screen of Candidate HP-Ag for Specificity and Off-TargetPotential

The candidate HP-Ag peptide sequences were then screened for peptidespecificity and off target reactivity potential using a BLASTp screenemploying parameters optimized for short sequence analysis andpreference for minimal substitution and compositional adjustments asspecificity for the intended target sequence is of utmost importance.Probability values for both On-target and Off-target returned resultsare then analyzed and a composite algorithm***-generated value is usedto determine an overall specificity rating. The greater the compositevalue the more specific the target sequence.

Using a similar approach to Step 1, analysis was first developedempirically and then an algorithm was designed for this evaluation toprovide consistency and reduce potential bias.

Candidate HP-Ag sequences that passed with high specificity and lowoff-target potential were qualified as HP-Ag (Table 4).

TABLE 4  HP-Ag sequences identified in the BRD4-NUT fusion region Speci-ficity Rating (Fold Difference between Specific SEQ Target and Quali-Candidate HP sequence ID  Non- fied (HLA Specificity) NO: Target) HP-Ag?TLRELERYV (A2) 27 1.33E+03 Yes ALPGPDMSM (A2, B15) 31 5.11E+03 YesMSMKPSAAL (A2, B7, B8) 32 1.87E+03 Yes CLSGAGAGK (A3, A11) 38  9.1E+02Yes VIAGSSKMK (A3, A11) 39  1.9E+03 Yes KPQAEKVDV (B7) 41 8.67E+02 YesSPALPFLPP (B7) 44 4.24E+03 Yes PPQPIMPSV (B7) 45 2.13E+03 YesAPGTPCGGL (B7) 46 2.28E+03 Yes GPAPPFVTA (B7) 47 6.76E+02 YesAGAGKVIVK (A3, A11) 200 6.70E+02 Yes NVKTILPSK (A3, A11) 201 6.79E+02Yes LVPIVPLEK (A3, A11) 202 5.77E+02 Yes IEIDFETLK (A11) 203 2.12E+03Yes PQPIMPSVF (A24, B15) 209 5.07E+02 Yes VFSPDNPLM (A24) 210 4.80E+03Yes FSPDNPLML (A24) 211 4.86E+03 Yes VTASNVKTI (A24) 213 1.93E+03 YesISKDVYENF (A24, B15) 214 2.23E+03 Yes SVESPDNPL (B7) 215 1.27E+03 YesPPVAQLVPI (B7) 216 1.65E+03 Yes RQWQRYKAL (B7, B8, B15) 218 1.07E+04 YesLERYVTSCL (B8) 219 2.86E+03 Yes CLRKKRKPQ (B8) 220 1.07E+03 YesRKKRKPQAE (B8) 222 7.40E+02 Yes NFILTQTAL (B8) 223 1.79E+03 YesTQTALNSTA (B15) 225 9.40E+02 Yes GLEGPAPPF (B15) 226 7.61E+02 YesRSKISKDVY (B15) 227 3.29E+03 Yes WQRYKALAR (B15) 228 5.91E+03 YesAEPSQTQNF (A24) 229 2.89E+03 Yes EIEIDFETL (A24) 230 1.64E+03 YesYKALARRHL (B8) 234 2.27E+03 Yes

Example 4 HP-Ag Peptides Homologous to Sequences within the FusionRegion of ALK Fusion Proteins Expressed in Cancer

Anaplastic lymphoma kinase (ALK) was first discovered as part of thefusion protein NPM-ALK in anaplastic large cell lymphoma. ALK fusionproteins have been recognized as oncogenic and the constitutive ALKactivity caused by ALK translocations is a current target of severalcancer drugs that block ALK activity. The predominant ALK fusionproteins are NPM-ALK, EML4-ALK and TMP3-ALK as well as additional lessfrequent translocations. However, normal ALK expression is seen inneural development and it remains at a low level in the adult brain.Also, ALK has a 64% homology to leukocyte tyrosine kinase (Turner et al.Leukemia 19:1128-1134 (2005)) and it belongs to the insulin receptorsuperfamily (Mourali et al. Molecular and Cellular Biology 26:6209-6222(2006)). These facts could place safe targeting of ALK by ACT out ofreach. These studies were conducted based on the hypothesis that ALKpositive tumors could be targeted for HP-ACT by specifically targetingthe novel sequence formed by the fusion. Of particular interest was alinker region shared by the ALK fusion proteins. Identifying specificantigenic sequences within this region would make ALK positive cancersfeasible indications for ACT therapy, in particular, HP-ACT.

The first step in these studies was evaluating the fusion protein forits target potential based on the parameters of frequency, pattern ofexpression, and its clinical and commercial feasibility (Frequency), itsability to discriminate cancer cells from normal cells (Specificity),and the strength of its functional relationship to the cancer's abilityto perpetuate itself (Functional Connectivity).

Step 1. Qualification of ALK Fusion Family Members as HP-TP or Aux-TP

A. TP Frequency

The first step was performed based on the hypothesis that suitable HP-Agneoantigens might be present within the novel fusion regions of the ALKfusion proteins that would not only allow the targeting of a specificALK fusion but also be applicable to several translocations and theirisoforms within the ALK fusion family. This would allow safe targetingof ALK by ACT while being able to use a target to treat multiple ALKpositive cancers. A sequence region was found that was shared bymultiple ALK fusions including EML4-ALK, NPM-ALK and TMP-ALK:

(SEQ ID NO: 50) KGAEIKTTNEVVLAVEFHPTDANTIITCGKSHIFFWTWSGNSLTRKQGIFGKYEKPKFVQCLAFLGNGDVLTGDSGGVMLIWSKTTVEPTPGKGPKGVYQLSKQLKAHDGSVFTLCQMRNGMLLTGGGKDRKIILWDHDLNPEREIMELQSPEYKLSKLRTSTIMTDYNPNYCFAGKTSSISDLKEVPRKNITLIRGLGHGAFGEVYEGQVSGMPNDPSPLQ.

Overall, EML4-ALK frequency in non-small cell lung cancer has beenreported at 4-13% (Shaw et al c

(2014); Shaw et al. J. Clinical Oncology 27(26):4247-4253 (2014)). Workon ALK drug targeting has helped define a subset of patients where thefrequency of EML4-ALK rises to 22% for patients with a history of littleto no smoking (Shaw et al. J. Clinical Oncology 27(26):4247-4253 (2014))and climbs to 33% for patients that do not have a mutation in epidermalgrowth factor receptor (EGFR mutations are present approximately 22% ofNSCLC) (Shaw et al. J Clinical Oncology 27(26):4247-4253 (2014)).According to SEER statistics, there are over 400,000 patients with lungcancer in the US alone, with an estimated 224,210 new cases and 159,260deaths expected in 2014. Even 4% of these numbers was sufficient toqualify EML4-ALK based on number of patients. Feasibility is increasedby the ability to triage the large patient population. Also, EML4-ALKmay be applicable to additional indications, which would furtherincrease its value. NPM-ALK is present in approximately 43% ofanaplastic large cell lymphoma stratified by age to as high as 83% inpediatric patients compared to 31% in adults. The high frequency withinALCL qualifies it as a feasible target for ACT in this indication.

One example of an indication that might not achieve a feasible frequencyon its own is the rare inflammatory myofibroblastic tumor (IMT). IMTsrepresent about 1% of lung tumors and it is estimated that up to 50% ofIMTs will be TMP3-ALK positive. Of note is that IMT can occur anywherein the body. While IMT is more common in the lung in young patients, ithas been reported in people of all ages (Gleason et al. J. Clin. Pathol.61:428-437 (2008)). Although these tumors have a low metastaticpotential, recurrence can be as high as 40% attributed to the lack ofability to entirely remove the tumor. IMT has been historicallydescribed using a number of terms, making its total prevalence difficultto estimate.

B. TP Specificity

Nucleophosmin (NPM) is a ubiquitous ‘housekeeping’ protein involved inmany basic cell functions including DNA replication, protein formationand cell cycle progression. Targeting epitopes common to normal NPMwould not be feasible. The same is true of the other ALK fusionpartners; echinoderm microtubule-associated protein like protein 4(EML4), binds and stabilizes mictotubules, the third major fusionpartner tropomyosin 3 is a normal component of the cytoskeleton. Allthree are important for normal cell function and so the fusion of ALKnow under their regulation drives constitutive ALK activity. Normalanaplastic lymphoma kinase (ALK) is more tightly expressed. In mice itappears during neural development and then remains in low amounts in theadult nervous system. In humans, ALK is detected in some pericytes (thecontractile cells of the microvasculature throughout the body) and inglia in some areas of the brain (Passoni et al. Blood 99:2100-2106(2002)). Both NPM-specific regions and ALK-specific regions will lackthe specificity needed to qualify it as an HP-TP candidate. However, ALKfusions are specific to cancer and rare disease. Targeting the fusionregion allows selective targeting of cells containing the abnormal ALKfusion while avoiding cells with normal NPM and ALK expression givingthe fusion protein a positive specificity value, if the antigen iswithin the unique region particular to the fusion protein.

C. TP Functional Connectivity

ALK has been shown to be a powerful driver of oncogenesis. Theexpression of ALK is driven by the fusion partner so the different ALKfusions exhibit preferential cancer expression for example: NPM-ALK inanaplastic lymphoma kinase; EML4-ALK in non-small cell lung cancer;TMP3-ALK in inflammatory myofibroblastic tumors. In all cases, thefusion results in constitutive expression of ALK. It acts through atleast three pathways with many interconnections: The Ras-ERG pathway,well-established as a driver of cell-cycle progression, the JAK-STAT andSTAT 3 pathways, involved in proliferation and survival respectively,and PI3K involved in survival and proliferation (Chiarle et al. NatureReviews Cancer 8:11-23 (2008)). More recently, NPM-ALK has beenconnected to increased Sox2 expression, Sox2 an important stem cellprotein involved in the maintenance of pluripotency in normal stem cells(Gelebart et al. Blood Cancer J. 2:e82; doi:10.1038/bcj.2012.27 (2012)).ALK is normally a transmembrane protein however the fusion renders itcytoplasmic, eliminating it as a candidate for CAR ACT. Since ALKactivity acts as a pivotal driver in ALK cancers, the likelihood thatC-RC would have to contain the fusion protein is high (Passoni et al.,Blood 99:2100-2106 (2002)) and the chance that cells lacking the fusionprotein would be C-RC in an ALK-fusion positive cancer is low. Thedependence on ALK activity afforded by the translocation established apositive connection to the C-RC of the cancer. Cells lacking expressionof the ALK fusion would be unlikely to perpetuate the cancer.

Curated analysis qualified the family of ALK fusion proteins as HP-TPand continuation to Step 2.

Step 2. Identification of Candidate HP-Ag Sequences

This example is not the first attempt to identify ALK T cell antigenssuitable for cancer immunotherapy and so in addition to identifyingfusion region antigens, Step 2 as disclosed herein was tested againstthe previous derivation of ALK fusion epitopes. In 2002, Passoni et al.(Passoni et al. Blood, 99:2100-2106 (2002)) identified several potentialT cell antigens to target abnormal ALK activity in anaplastic lymphomakinase that harbors an NPM-ALK translocation. The Passoni strategy wasto avoid the ubiquitous NPM and focus on the more restricted anddifferentially expressed ALK. ALK-specific targeting will haveinsufficient specificity to qualify ALK kinase-region antigens forHP-ACT, making peptides from the ALK kinase region unsuitable for HP-ACTdevelopment. This experiment aimed to compare the Passoni method ofepitope identification with the method of Step 2 as disclosed herein, intheir ability to discern T reactive epitopes. The ability to predict the9 amino acid core sequences identified by Passoni. using step 2 asdisclosed herein was assessed.

Passoni began their studies by assessing potential binding of ALKpeptides using a single method that estimated binding to HLA A2, andselecting 22, 9 and 10 amino acid peptides within and bordering thekinase region of ALK. Passoni then tested the peptides for their abilityto mount a response in transgenic mice as well as in vitro, usingtransgenic mouse lymphocytes and naïve normal human donor lymphocytes.Of the 22 predicted peptides, 9 exhibited strong binding to HLA A2 withsufficient stability to likely elicit a T cell response. In vivo, 7 ofthe 9 peptides were able to mount a T cell response in mice transgenicfor HLA A2. Differences in outcome emphasized that affinity alonewithout sufficient stability was an ineffective predictor of T cellresponse. They identified two 10 amino acid peptides that were capableof stimulating a T cell response in transgenic mice, killing of NPM-ALKpositive cells, and that could stimulate T cells from one of threenormal patients.

The selection process disclosed herein factors in affinity and stabilityas well as other parameters for more efficient identification ofpotential epitopes. Step 2 was able to identify core 9mers within the10mer antigens with some important additional information. Of the 9mersequences within the 22 peptides selected by Passoni Step 2 would haveeliminated 7 epitopes before T cell selection and would have identifiedall 9 positive responders for T cell screening. Of the 9 reactivepeptides, Passoni ultimately identified SLAMLDLLHV (SEQ ID NO:51) andGVLLWEIFSL (SEQ ID NO:52) as reactive human T cell antigens. Step 2identified LAMLDLLHV (SEQ ID NO:53) and VLLWEIFSL (SEQ ID NO:54) as highprobability epitopes and therefore would have selected for core 9 aminoacid sequences within the peptides selected as best by Passoni. However,within GVLLWEIFSL (SEQ ID NO:52), Step 2 predicted VLLWEIFSL (SEQ IDNO:54) to be a very strong epitope whereas GVLLWEIFS was not. This issupported by Passoni's own data which showed that transgenic animalsimmunized with the VLLWEISFSL peptide generated HLA A2 T cells thatexhibited better T cell lysis (E/T ratio of 48-24-21) than miceimmunized with GVLLWEIFSL (SEQ ID NO:52) (E/T ratio of 24-15-15). WithinSLAMLDLLHV (SEQ ID NO:51), the SLAMLDLLH (SEQ ID NO:199) 9mer did notqualify as an epitope in these studies, although LAMLDLLHV (SEQ IDNO:53) did, again suggesting that the reactivity was more dependent onthe C-terminal portion of the peptide. This provides evidence that theStep 2 screen is able to capture high probability T cell epitopes withgreater efficiency and predictability while providing additionalinformation that can aid the use of the sequences as tools for T cellselection and ACT design.

While Passoni believed that they had to avoid targeting NPM because ofits ubiquitous nature, they believed that ALK cross-reactivity would benon-existent. However, recent clinical experience in the use of MAGE A3(Melanoma-associated antigen 3) targets for ACT (a target noted byPassoni as support for the safety of such targets back in 2002), make itclear that ALK itself is unlikely to be a feasible target for ACTdespite its natural antigenicity. This barrier to ALK fusions as an ACTtarget is eliminated by focusing on the shared fusion region of themajor ALK fusion proteins and their isoforms.

The shared fusion region:KGAEIKTTNEVVLAVEFFHPTDANTIITCGKSHIFFWTWSGNSLTRKQGIFGKYEKPKFVQCLAFLGNGDVLTGDSGGVMLIWSKTTVEPTPGKGPKGVYQLSKQLKAHDGSVFTLCQMRNGMLLTGGGKDRKIILWDHDLNPEREIMELQSPEYKLSKLRTSTIMTDYNPNYCFAGKTSSISDLKEVPRKNITLIRGLGHGAFGEVYEGQVSGMPNDPSPLQ (SEQ ID NO:55) was used for the discoveryof shared HP-Ag peptides. Bold indicates sequence shared by EML4-ALKisoforms, NPM-ALK and TMP3 ALK.

Overlapping 9 amino acid sequences within the fusion region wereevaluated manually by valuing the sequence's chemistry, antigenprocessing, HLA specificity, and binding kinetics. A total of 212peptides were analyzed. Several sequences stood out as having comparablemolecular characteristics as good or better than well-characterizedepitopes with known in vivo immunogenicity and in particular, T cellreactivity. The system was developed using HLA A2 as the model but mostcommon HLA types could be analyzed. Major HLA types were chosen, thatwould represent a majority of patients in major populations.

High Probability ALK fusion region sequences with their HLA specificityare shown in Table 5.

TABLE 5  High Probability ALK fusion region sequences(candidate HP-Ag sequences) with their HLA specificity HLA Core 9mer SEQTarget (s) Specificity sequence ID NO: EML4-ALK A2 TTNEVVLAV 56 EML4-ALKA2 VLAVEFHPT 57 EML4-ALK A2, A24 KFVQCLAFL 58 EML4-ALK A2 FLGNGDVLT 59EML4-ALK A2 VLTGDSGGV 60 EML4-ALK A2 MLIWSKTTV 61 EML4-ALK A2 KIILWDHDL62 EML4-ALK A2 ILWDHDLNP 63 EML4-ALK; NPM-ALK;  A2 ELQSPEYKL 64 TMP3-ALKEML4-ALK A2 GMPNDPSPL 65 EML4-ALK A3 WSGNSLTRK 66 EML4-ALK A3 TTVEPTPGK67 EML4-ALK A3 SVFTLCQMR 68 EML4-ALK A3 GMLLTGGGK 69 EML4-ALK; NPM-ALK; A3 RTSTIMTDY 70 TMP3-ALK EML4-ALK; NPM-ALK;  A3 IMTDYNPNY 71 TMP3-ALKEML4-ALK; NPM-ALK;  A3 KTSSISDLK 72 TMP3-ALK EML4-ALK; NPM-ALK;  A3ITLIRGLGH 73 TMP3-ALK EML4-ALK B7 HPTDANTII 74 EML4-ALK B7 KPKFVQCLA 75EML4-ALK B7 TPGKGPKGV 76 EML4-ALK B7 NPEREIMEL 77 EML4-ALK; NPM-ALK;  B7SPEYKLSKL 78 TMP3-ALK EML4-ALK; NPM-ALK;  B7 VPRKNITLI 79 TMP3-ALKEML4-ALK A24 AFLGNGDVL 80 EML4-ALK A24 CQMRNGMLL 81 EML4-ALK; NPM-ALK; A24 CFAGKTSSI 82 TMP3-ALK EML4-ALK A11 TTNEVVLAV 56 EML4-ALK A11WSGNSLTRK 66 EML4-ALK A11 GGVMLIWSK 235 EML4-ALK A11 TTVEPTPGK 67EML4-ALK A11 VYQLSKQLK 236 EML4-ALK A11 SVFTLCQMR 68 EML4-ALK A11GMLLTGGGK 69 EML4-ALK A11 LTGGGKDRK 237 EML4-ALK; NPM-ALK;  A11QSPEYKLSK 238 TMP3-ALK EML4-ALK; NPM-ALK;  A11 RTSTIMTDY 70 TMP3-ALKEML4-ALK; NPM-ALK;  A11 KTSSISDLK 72 TMP3-ALK EML4-ALK; NPM-ALK;  A11ISDLKEVPR 239 TMP3-ALK EML4-ALK B8 EIKTTNEVV 240 EML4-ALK B8 NSLTRKQGI241 EML4-ALK B8 SLTRKQGIF 242 EML4-ALK B8 YEKPKFVQC 243 EML4-ALK B8QLKAHDGSV 244 EML4-ALK B8 LCQMRNGML 245 EML4-ALK B8 GGKDRKIIL 246EML4-ALK; NPM-ALK;  B8 SPEYKLSKL 78 TMP3-ALK EML4-ALK; NPM-ALK;  B8LSKLRTSTI 247 TMP3-ALK EML4-ALK; NPM-ALK;  B8 CFAGKTSSI 82 TMP3-ALKEML4-ALK; NPM-ALK;  B8 EVPRKNITL 248 TMP3-ALK EML4-ALK; NPM-ALK;  B8VPRKNITLI 79 TMP3-ALK EML4-ALK B15 ITCGKSIHF 249 EML4-ALK B15 SLTRKQGIF242 EML4-ALK B15 MLIWSKTTV 61 EML4-ALK B15 QLKAHDGSV 244 EML4-ALK B15LKAHDGSVF 249 EML4-ALK B15 CQMRNGMLL 81 EML4-ALK; NPM-ALK;  B15IMELQSPEY 34 TMP3-ALK EML4-ALK; NPM-ALK;  B15 RTSTIMTDY 70 TMP3-ALKEML4-ALK; NPM-ALK;  B15 IMTDYNPNY 71 TMP3-ALK

Step 3. Screen of Candidate HP-Ag for Specificity and Off-TargetPotential

The selected peptide sequences were then screened for peptidespecificity and off target reactivity potential using a BLASTp screenemploying parameters optimized for short sequence analysis andpreference for minimal substitution and compositional adjustments asspecificity for the intended target sequence is of utmost importance.Probability values for both On-target and Off-target returned resultsare then analyzed and a composite algorithm-generated value is used todetermine an overall specificity rating. The greater the composite valuethe more specific the target sequence.

Using a similar approach to Step 1, analysis was first developedempirically and then an algorithm was designed for this evaluation toprovide consistency and reduce potential bias.

Candidate HP-Ag sequences that passed with high specificity and lowoff-target potential were qualified as HP-Ag (Table 6).

TABLE 6  HP-Ag sequence identified in EML4-ALK SpecificityRating Assessed Candidate HP Fold Difference sequence between SEQ (HLASpecific Target Qualified ID Specificity) and Non-Target HP-Ag? NO:TTNEVVLAV (A2) (A11) 6.55E+02 Yes 56 VLAVEFHPT (A2) 9.53E+02 Yes 57KFVQCLAFL (A2, A24) 1.47E+03 Yes 58 FLGNGDVLT (A2) 1.24E+03 Yes 59MLIWSKTTV (A2), (B15) 1.60E+04 Yes 61 KIILWDHDL (A2) 1.54E+04 Yes 62ILWDHDLNP (A2) 7.00E+03 Yes 63 *ELQSPEYKL (A2) 1.70E+03 Yes 64GMPNDPSPL (A2) 3.67E+03 Yes 65 WSGNSLTRK (A3), (A11) 4.35E+03 Yes 66TTVEPTPGK (A3), (A11) 1.10E+03 Yes 67 SVFTLCQMR (A3), (A11) 1.24E+03 Yes68 *RTSTIMTDY (A3),  1.57E+04 Yes 70 A11, B15) *IMTDYNPNY (A3)(B15)1.95E+04 Yes 71 *ITTIRGLGH (A3) 6.95E+02 Yes 73 HPTDANTII (B7) 9.22E+02Yes 74 KPKFVQCLA (B7) 9.33E+02 Yes 75 *SPEYKLSKL (B7)(B8) 2.25E+03 Yes78 *VPRKNITLI (B7)(B8) 3.08E+03 Yes 79 AFLGNGDVL (A24) 1.57E+03 Yes 80*CQMRNGMLL (A24)(B15) 2.25E+04 Yes 81 *CFAGKTSSI (A24)(B8) 2.29E+03 Yes82 GGVMLIWSK (A11)  4.2E+03 Yes 235 LTGGGKDRK (A3, A11) 6.44E+02 Yes 237QSPEYKLSK (A3, A11) 2.68E+03 Yes 238 *ISDLKEVPR (A11) 9.33E+02 Yes 239EIKTTNEVV (B8) 1.17E+03 Yes 240 NSLTRKQGI (B8) 6.71E+02 Yes 241SLTRKQGIF (B8)(B15) 9.99E+02 Yes 242 YEKPKFVQC (B8) 1.15E+03 Yes 243LCQMRNGML (B8) 2.25E+04 Yes 245 *EVPRKNITL (B8) 3.02E+03 Yes 248ITCGKSHIF (A24)(B15) 2.25E+03 Yes 249 *also identified in NPM-ALK;TMP3-ALK

The following sequences did not qualify as HP-Ag: SEQ ID NO: 34 (alsoidentified in NPM-ALK; TMP3-ALK); SEQ ID NO:35; SEQ ID NO:60; SEQ IDNO:69; SEQ ID NO:72, SEQ ID NO:76; SEQ ID NO: 77; VYQLSKQLK (A11, A24)(SEQ ID NO: 236); QLKAHDGSV (B8)(B15) (SEQ ID NO:244; GGKDRKIIL (B8)(SEQ ID NO:246); LSKLRTSTI (B8) (SEQ ID NO:247) (also identified inNPM-ALK; TMP3-ALK); and LKAHDGSVF (B15) (SEQ ID NO:309).

Example 5 HP-Ag Peptides with Sequence Homology to the Fusion Region ofTMPRSS2-ERG Expressed in Prostate Cancer

The potential of the TMPRS52-ERG as an HP-TP was evaluated using curatedliterature research as well as data from protein and genome databases.

Step 1. Qualification of TMPRSS2-ERG as HP-TP or Aux-TP

A. TP Frequency

Translocations of the ERG gene have resulted in several different fusionproteins in addition to TMPRSS2-ERG: EWS-ERG in Ewing's sarcoma andFUS-ERG in myeloid leukemia as well as NDRG1-ERG in prostate cancer. ETSfusions rank third in all advanced prostate cancer mutations and over80% are ERG fusions (Robinson et al. Cell 161:1215 (2015)). TheTMPRSS2-ERG fusion pair is present on average in approximately 50% ofall prostate cancers. This qualifies it for frequency.

B. TP Specificity

The fusion gene is abnormal and will not be present in normal cellsgiving the target a high specificity.

C. TP Functional Connectivity

ERG (ETS-regulated gene), an erythroblast transformation-specific

(ETS) transcription factor is abnormally upregulated by thetranslocation and fusion. Notably, ETS family members are associatedwith embryonic development, cell proliferation and differentiation (Genecards). TMPRSS2 (transmembrane protease, serine 2) expression is higheror lower depending on the stage of prostate cancer and may not bepivotal in all stages of prostate cancer. ERG was then evaluated for itssignificance to prostate cancer biology. ERG's inherent function hasbeen linked with self-renewal (Casey et al. PLoS One 7(7):e41668(2012)). There is evidence that ERG promotion of self-renewal can fuelthe accumulation of additional mutations in the proliferative cellcompartment and eventually some mutations may overcome the need for ERGexpression, even in some TMPRSS2-ERG containing cancers. However, morerecent clinical data on expression of the fusion protein in metastasessuggest excellent retention of the fusion protein's expression inmetastatic disease (Robinson et al. Cell 161:1215 (2015)).

Step 1 directs the use of this target toward cancers where ERG-drivenself-renewal is still a factor in the cancer's regeneration andestablishes the potential relative value of the target as a treatmentearly in the process so that the potential targets are neither missednor improperly properly used.

Step 2. Identification of Candidate HP-Ag Sequences

The TMPRSS2-ERG fusion region sequence used was:

(SEQ ID NO: 83) MTASSSSDYGQTSKMSPRVPQQDWLSQPPARVTIKMECNPSQVNGSRNSPDECSVAKGGKMVGSPDTVGMNYGSYMEEKHMPPPNMTTNERRVIVPADPTLWSTDHVRQWLEWAVKEYGLPDVNILLFQNIDGKELCKMTKDDFQRLTPSYNADILLSHLHYLRETPLPHLTSDDVDKALQNSPRLMHARNTGGAAFIFPNTSVYPEATQRITTRPVSYRA total of 212 overlapping 9 amino acid sequences were analyzed for eachHLA type shown and relevant sequences identified (Table 7).

TABLE 7 Candidate HP-Ag sequences in TMPRSS2-ERGfusion region with their HLA specificity HLA Core 9 mer SEQ TargetSpecificity sequence ID NO: TMPRSS2-ERG A2 WLSQPPARV  84 TMPRSS2-ERG A2KMVGSPDTV  85 TMPRSS2-ERG A2 VIVPADPTL  86 TMPRSS2-ERG A2 GLPDVNILL  87TMPRSS2-ERG A2, A24, B8 ILLSHLHYL  88 TMPRSS2-ERG A2 KMECNPSQV  89TMPRSS2-ERG B7, B8 KALQNSPRL  90 TMPRSS2-ERG A3, A11 TLWSTDHVR  91TMPRSS2-ERG A3, B27, A11 RQWLEWAVK  92 TMPRSS2-ERG A3, A11 LLFQNIDGK  93TMPRSS2-ERG A3, A11 NIDGKELCK  94 TMPRSS2-ERG A3, A11 KMTKDDFQR  95TMPRSS2-ERG A3, A11 LLSHLHYLR  96 TMPRSS2-ERG A3, A11 HLTSDDVDK  97TMPRSS2-ERG A3, B15 FIFPNTSVY  98 TMPRSS2-ERG A3, A11 SVYPEATQR  99TMPRSS2-ERG A3, B15 RITTRPVSY 100 TMPRSS2-ERG A3, A11 ITTRPVSYR 101TMPRSS2-ERG A24 EYGLPDVNI 102 TMPRSS2-ERG A24 VYPEATQRI 103 TMPRSS2-ERGB7 SPRVPQQDW 104 TMPRSS2-ERG B7 PPARVTIKM 105 TMPRSS2-ERG B7 LPDVNILLF106 TMPRSSZ-ERG B7 TPSYNADIL 107 TMPRSS2-ERG B7 LPHLTSDDV 108TMPRSS2-ERG B7 HARNTGGAA 109 TMPRSS2-ERG B7, B15 YPEATQRIT 110TMPRSS2-ERG B27 PRVPQQDWL 111 TMPRSS2-ERG B27 ARVTIKMEC 112 TMPRSS2-ERGB27 RRVIVPADP 113 TMPRSS2-ERG B27 VRQWLEWAV 114 TMPRSS2-ERG B27QRLTPSYNA 115 TMPRSS2-ERG B27 LRETPLPHL 116 TMPRSS2-ERG B27 ARNTGGAAF117 TMPRSS2-ERG A3, A11 SSDYGQTSK 250 TMPRSS2-ERG A11, B15 MTASSSSDY 251TMPRSS2-ERG A11 GQTSKMSPR 252 TMPRSS2-ERG A24 SQPPARVTI 253 TMPRSS2-ERGA24 NYGSYMEEK 254 TMPRSS2-ERG A24 SYMEEKHMP 255 TMPRSS2-ERG A24VNILLFQNI 256 TMPRSS2-ERG A24 HYLRETPLP 257 TMPRSS2-ERG A24 NTGGAAFIF258 TMPRSS2-ERG B7 VPQQDWLSQ 259 TMPRSS2-ERG B7 VPADPTLWS 260TMPRSS2-ERG B7 SPRLMHARN 261 TMPRSS2-ERG B8 MTKDDFQRL 262 TMPRSS2-ERG B8LHYLRETPL 263 TMPRSS2-ERG B15 LQNSPRLMH 264 TMPRSS2-ERG B15 TVGMNYGSY265 TMPRSS2-ERG B15 WLEWAVKEY 266 TMPRSS2-ERG B15 FQNIDGKEL 267TMPRSS2-ERG B15 TQRITTRPV 268

Step 3. Screen of Candidate HP-Ag Sequences for Specificity andOff-Target Potential

The selected peptide sequences were then screened for peptidespecificity and off target reactivity potential using a BLASTp screenemploying parameters optimized for short sequence analysis andpreference for minimal substitution and compositional adjustments asspecificity for the intended target sequence is of utmost importance.Probability values for both On-target and Off-target returned resultsare then analyzed and a composite algorithm-generated value is used todetermine an overall specificity rating. The greater the composite valuethe more specific the target sequence.

Using a similar approach to Step 1, analysis was first developedempirically and then an algorithm was designed for this evaluation toprovide consistency and reduce potential bias.

Candidate HP-Ag sequences that passed with high specificity and lowoff-target potential were qualified as HP-Ag (Table 8).

TABLE 8 HP-Ag sequences in TMPRSS2-ERG fusion regionAssessed Fold Difference Candidate HP sequence between Specific TargetQualified SEQ (HLA Specificity) and Non-Target HP-Ag? ID NO:WLSQPPARV (A2) 4.23E+03 Yes  84 VIVPADPTL (A2) 1.74E+03 Yes  86GLPDVNILL (A2) 8.00E+02 Yes  87 ILLSHLHYL (A2, A24, B8) 1.04E+03 Yes  88KMECNPSQV (A2) 1.11E+04 Yes  89 KALQNSPRL (B7, B8) 1E+03 Yes  90TLWSTDHVR (A3, A11) 3.24E+03 Yes  91 RQWLEWAVK (A3, A11, B27) 2.00E+03Yes  92 LLFQNIDGK (A3, A11) 7.00E+02 Yes  93 KMTKDDFQR (A3, A11)2.91E+03 Yes  95 LLSHLHYLR (A3, A11) 6.40E+02 Yes  96HLTSDDVDK (A3, A11) 1.45E+03 Yes  97 FIFPNTSVY (A3, B15) 7.13E+02 Yes 98 FIFPNTSVY RITTRPVSY (A3, A11, B15) 4.46E+03 Yes 100 EYGLPDVNI (A24)1.95E+03 Yes 102 VYPEATQRI (A24) 1.43E+03 Yes 103 SPRVPQQDW (B7)7.80E+03 Yes 104 PPARVTIKM (B7) 3.50E+03 Yes 105 TPSYNADIL (B7) 1.88E+03Yes 107 HARNTGGAA (B7) 1.54E+03 Yes 109 YPEATQRIT (B7) 4.50E+03 Yes 110PRVPQQDWL (B27) 9.18E+03 Yes 111 ARVTIKMEC (B27) 6.67E+03 Yes 112RRVIVPADP (B27) 1.75E+03 Yes 113 QRLTPSYNA (B27) 1.33E+03 Yes 115ARNTGGAAF (B27) 1.62E+03 Yes 117 SSDYGQTSK (A3, A11) 1.49E+03 Yes 250GQTSKMSPR (A11) 2.56E+03 Yes 252 SQPPARVTI (A24) 1.47E+03 Yes 253NYGSYMEEK (A11, A24) 5.72E+03 Yes 254 SYMEEKHMP (A24) 3.62E+04 Yes 255VNILLFQNI (A24) 1.14E+03 Yes 256 HYLRETPLP (A24) 2.43E+03 Yes 257NTGGAAFIF (A24) 4.17E+03 Yes 258 VPQQDWLSQ (B7) 6.00E+03 Yes 259VPADPTLWS (B7) 3.18E+03 Yes 260 SPRLMHARN (B7) 7.40E+03 Yes 261MTKDDFQRL (B8) 7.59E+03 Yes 262 LHYLRETPL (B8) 2.90E+03 Yes 263LQNSPRLMH (B5) 4.91E+03 Yes 264 TVGMNYGSY (B15) 8.75E+03 Yes 265WLEWAVKEY (B15) 2.78E+04 Yes 266 FQNIDGKEL (B15) 1.47E+03 Yes 267

Example 6 HP-Ag Peptides Homologous to Sequences within the CancerTestis Antigen A-Kinase Anchor Protein 4 (AKAP4 (AKAP82, Cancer TestisAntigen 99))

Cancer testis antigen AKAP4 is highly restricted to the sperm's fibroussheath. It is essential for sperm motility (Hu). However, AKAP 4 hasbeen reported to be widely and stably expressed in several human cancersmaking it a cancer biomarker and a potential candidate for ACT. Thepotential of AKAP4 as a target for cancer diagnostics as well as cancerimmunotherapy, including adoptive immunotherapy has been recognized byothers (Chiriva-Internati et al. The Prostate 72(1):12-23 (2012); US2012/0263757 A1; WO2014127006A1)),) though not necessarily to target theC-RC nor with any delineation of specific peptide antigens or theirqualification. Identification of specific peptide epitopes isparticularly important for ACT since AKAP4 is part of a larger family ofAKAPs expressed in adult tissues. For its use in HP-ACT, manipulationmust be at the level of the T cell (the most direct and robust mode ofimmune manipulation). AKAP4 has to qualify as an HP-TP or Aux-TP (Step1), and HP-Ag sequences must be identified and qualified for HP-ACTdevelopment (Steps 2-3).

Step f. Qualification of AKAP4 as an HP-TP or Aux-TP

A. TP Frequency

In a survey of AKAP4 expression in breast cancer specimens, Saini et al.(Saini et al. PLoS One 8(2):e57095 (2013)) found the protein expressedin 85% of breast cancer specimens regardless of stage, type and grade ofthe tumor. AKAP4 was also found in 89% of ovarian cancer specimensregardless of stage (Agarwal et al. OncoImmunology 2(5):e24270 (2013)).Its expression has also been described in cervical (Agarwal et al. Int.J Gynecol. Cancer 23(4):650-658 (2013)), prostate (Chiriva-Internati etal. The Prostate 72(1):12-23 (2012)) and possibly non-small cell lungcancers (Rhadi et al. J. Clin. Oncol. 31 suppl:abstr e18527 (2013)).AKAP4 protein has also been found in multiple myeloma (Chiriva-Intematiet al. Br. J. Haematol. 140:464-474 (2008)). AKAP4's high frequency ofexpression, independent of stage in at least two cancers, and itspresence in multiple cancers gives it a high frequency value.

B. TP Specificity

Although there are many forms of AKAPs functioning in normal tissues,normal AKAP4 expression is specific to the sperm's fibrous sheath. It isa highly conserved protein across species indicating a very specific andspecialized normal function. In cancerous lesions, AKAP4 expression isrestricted to the cancer cells of the tumor and is not observed in thesurrounding cells (Agarwal et al. OncoImmunology 2(5):e24270 (2013)).Tight, conserved normal expression and highly delimited expression incancer patients contribute to a high Specificity Value for AKAP4.

C. TP Functional Connectivity

What was known about AKAP4 and its similar family member AKAP110 wasused to determine whether AKAP4 qualified as a cancer driver that couldhave a pivotal connection to the propagation of AKAP4⁺ cancers. As aclass of proteins, AKAPs hold protein kinase (PKA), the principalintracellular receptor for cyclic AMP (cAMP) and other signalingmolecules in proximity to specific substrates within the cell. In doingso they orchestrate PKA activity. It is known that the AKAPs governsubcellular targeting of PKA activity to specific cellular compartmentsand target substrates. They also bind additional signaling molecules.PKA has a multi-functional role in control of cell proliferation,survival and differentiation and is one of the most recognized driversof carcinogenesis.

AKAPs tether the PKA holoenzyme (a coenzyme and an apoenzyme), whichconsists of two regulatory subunits (R) and two catalytic subunits (C).AKAP RI and RII classes differ in their sensitivity to cAMP, pattern ofphosphorylation and subcellular localization. AKAP4 (AP85) is a memberof the AKAP110 family. Like AKAP110, AKAP4 has sites for both RIα andRIIα. It is known that AKAP110, a slightly larger family member thanAKAP4, has both cyclic AMP-dependent and cyclic AMP-independentmechanisms for PKA activation (Andreeva et al. J. Molecular Signaling2:13-21 (2007)). Therefore neoexpression of AKAP4 in somatic cellslikely provides more than one upstream mechanism (cAMP dependent andindependent) to disrupt PKA control.

AKAP4 exhibits abundant and broadly localized expression within cancercells both in vitro and in vivo. AKAP4 has been shown to associate withmicrotubules when artificially expressed in normal somatic cells (Nipperet al. Biology of Reproduction 75:189-196 (2006)) suggesting that it isbe capable of a broad intracellular distribution when abnormallyexpressed. Distribution of AKAP4 within cervical cancer cells wasassociated with mitochondria, golgi, the cytoplasm, as well as surfaceexpression. This further supports AKAP4's potential to disrupt normalcontrol of PKA. Mutated PKA is one of the most well-recognized andwell-characterized cancer drivers. However in the case of AKAP4 positivecancers, since the abnormality is upstream of PKA, PKA will no longerdrive the cancer in the absence of AKAP4. Experimental evidence for thisis that when AKAP4 is silenced in AKAP4 positive cervical cancer cellsin vitro, they lose colony forming ability, this ability being ahallmark of regeneration-capable cells. In cervical cancer cells andcell lines, colony forming ability was consistently slightly greaterthan 50% in the cancer cells, supporting its action in an albeitsubstantial subpopulation of the cancer cells. AKAP4 expression in tumorspecimens correlated well with PCNA, a marker of cell proliferation.Silencing of AKAP4 expression led to formation of small, slow growingtumors in mice with a fibrous morphology as opposed to those with activeAKAP4 that exhibited small epithelial morphology with high PCNAstaining. This lends further support to AKAP4's pivotal connection tothe propagation of epithelial cancer. Cells within AKAP4⁺ cancerslacking AKAP4 will be incapable of propagating the cancer. AKAP4'srestriction to cancer cells in vivo, as well as its stable expressionacross type and stage of a cancer supports its essential role.

There is recent clinical support to AKAP4's significance in lung cancer.Gumireddy et al. (Gumireddy et al. Oncotarget 6(19):1-11 (2015)).reported that of 116 cancer testis antigens screened for diagnosticpotential in 264 non-small cell lung cancer (NSCLC) patients and 135control patients, only AKAP4 predicted the presence, recurrence andprogression of NSCLC Its presence in the blood could distinguish betweenpatients with cancerous and benign lesions, detect recurrence of thecancer following surgery before a tumor was detected and predicted thesubsequent development of metastatic disease.

In addition to data mining of AKAP4 biochemistry and PKA action incancer, AKAP4's role in cancer regeneration, more specifically the C-RC,can be corroborated using in vitro techniques able to specificallyselect the C-RC population from human tumors for analysis andexperimental manipulation.

AKAP4 qualified as an HP-TP for multiple cancer indications.

Step 2. Identification of Candidate HP-Ag Sequences

Qualified as an HP-TP, AKAP4 advanced to Step 2 where the protein wasanalyzed for high probability T cell epitopes. The AKAP4 sequence usedfor epitope analysis:

(SEQ ID NO: 118) MNRPQNLRLEMTAAKNTNNNQSPSAPPAKPPSTQRAVISPDGECSIDDLSFYVNRLSSLVIQMAHKEIKEKLEGKSKCLHHSICPSPGNKERISPRTPASKIASEMAYEAVELTAAEMRGTGEESREGGQKSFLYSELSNKSKSGDKQMSQRESKEFADSISKGLMVYANQVASDMMVSLMKTLKVHSSGKPIPASVVLKRVLLRHTKEIVSDLIDSCMKNLHNITGVLMTDSDFVSAVKRNLENQWKQNATDIMEAMLKRINSALIGEEKETKSQSLSYASLKAGSHDPKCRNQSLEFSTMKAEMKERDKGKMKSDPCKSLTSAEKVGEHILKEGLTIWNQKQGNSCKVATKACSNKDEKGEKINASTDSLAKDLIVSALKLIQYHLTQQTKGKDTCEEDCPGSTMGYMAQSTQYEKCGGGQSAKALSVKQLESHRAPGPSTCQKENQHLDSQKMDMSNIVLMLIQKLLNENPFKCEDPCEGENKCSEPRASKAASMSNRSDKAFEQCQEHQELDCTSGMKQANGQFIDKLVESVMKLCLIMAKYSNDGAALAELEEQAASANKPNERGTRCIHSGAMPQNYQDSLGHEVIVNNQCSTNSLQKQLQAVLQWIAASQFNVPMLYFMGDKDGQLEKLPQVSAKAAEKGYSVGGLLQEVMKFAKERQPDEAVGKVARKQLLDWLLANL

A total of 678 overlapping 9 amino acid sequences (9mers) were screenedusing a comprehensive evaluation of antigenicity, chemistry,biochemistry, processing, and HLA binding. Five prevalent HLA A and HLAB types found in major world populations were screened for candidateepitopes and candidate sequences identified (Table 9).

TABLE 9 Candidate HP-Ag sequences in AKAP4 with their HLA specificityHLA Core 9 mer SEQ Target Specificity sequence ID NO: AKAP4 A2 SIDDLSFYV119 A2 YVNRLSSLV 120 A2, A3 B15 RLSSLVIQM 121 A2 GLMVYANQV 122 A2, B8MMVSLMKTL 123 A2, B8 VLLRHTKEI 124 A2 VLMTDSDFV 125 A2 AMLKRLVSA 126 A2KMDMSNIVL 127 A2, A24, B8 VLMLIQKLL 128 A2 YQDSLGHEV 129 A2 SLQKQLQAV130 A2 GQLEKLPQV 131 A2 LLDWLLANL 132 A2 VASDMMVSL 133 A2 LIDSCMKNL 134A2, B8 NLHNITGVL 135 A2 IMEAMLKRL 136 A2, B8, B15 MLKRLVSAL 137 A2KINASTDSL 138 A2 LIVSALKLI 139 A2, B8 ALKLIQYHL 140 A2 DMSNIVLML 141 A2IVLMLIQKL 142 A2 LLNENPFKC 143 A2 FIDKLVESV 144 A2, A3, B15 KLVESVMKL145 A2 ALAELEEQA 146 A2 QLQAVLQWI 147 A2, B8 FMGDKDGQL 148 A2 KLPQVSAKA149 A2, B8 KAAEKGYSV 150 A2 SVGGLLQEV 151 A2 LLQEVMKFA 152 AKAP4 A3, A11SLVIQMAHK 153 A3, A11 SICPSPGNK 154 A3, A11 FLYSELSNK 155 A3 KQMSQRESK156 A3 KEFADSISK 157 A3, B15 SISKGLMVY 158 A3, A11 MVSLMKTLK 159 A3, A11TLKVHSSGK 160 A3 VVLKRVLLR 161 A3 VLKRVLLRH 162 A3, A11 QSLSYASLK 163A3, A11 QSLEFSTMK 164 A3 HLTQQTKGK 165 A3 KCGGGQSAK 166 A3, A1 NIVLMLIQK167 A3, A11 KLLNENPFK 168 A3, B15 KLCLIMAKY 169 A3, A11, B15 SQFNVPMLY170 A3, A11 QVSAKAAEK 171 AKAP4 A24, B8 FYVNRLSSL 172 A24 KYSNDGAAL 173A24 QFNVPMLYF 174 AKAP4 A11 IQMAHKEIK 175 A11, A3 ISPRTPASK 176 A11KQMSQRESK 177 A11 VVLKRVLLR 178 A11 MAQSTQYEK 179 A3, A11 ASMSNRSDK 180A11 ASANKPNFR 181 A3, A11 QSPSAPPAK 182 AKAP4 B7, A24 RPQNLRLEM 183B7, A24 KPPSTQRAV 184 B7, A24 PPSTQRAVI 185 B7, B8, A24 SPRTPASKI 186B7, A24 KPIPASVVL 187 B7, 138, A24 DPKCRNQSL 188 B7, A24 CPGSTMGYM 189B7, B8, A24 MPQNYQDSL 190 B7, A24 LPQVSAKAA 191 AKAP4 B15, B7, B15CSIDDLSFY 192 B15 ETKSQSLSY 193 B15 SQSLSYASL 194 B15 NQSLEFSTM 195 B15GMKQANGQF 196 B8, B15 LQKQLQAVL 197 B15 LQWIAASQF 198 A3, A11 PIPASVVLK275 A3, A11 VSALIGEEK 276 A3, A11 NASTDSLAK 277 A3 KDLIVSALK 278 A3, A11QSAKALSVK 279 A3 KCSEPRASK 280 A3 ELDCTSGMK 281 A3, A11 QANGQFIDK 282A3, A11 QCSTNSLQK 283 A3, A11 RQPDEAVGK 284 A11 YSELSNKSK 285 A11SDMMVSLMK 286 A11 TDIMEAMLK 287 A11 FSTMKAEMK 288 A11 GNSCKVATK 289 A11EVMKFAKER 290 A24 VSAVKRNLF 291 A24, B7 APPAKPPST 292 A24, B7 EPRASKAAS293 B8 MNRPQNLRL 294 B8 NLRLEMTAA 295 B8 DLSFYVNRL 296 B8 KLEGKSKCL 297B8 SVVLKRVLL 298 B8 EAMLKRLVS 299 B8 EKETKSQSL 300 B8 VGKVARKQL 301 B15GVLMTDSDF 302 B15 ILKEGLTIW 303 B15 KLIQYHLTQ 304 B15 GLLQEVNMK 305

Step 3. Screen of Candidate HP-Ag Sequences for Specificity andOff-Target Potential

The selected peptide sequences were then screened for peptidespecificity and off target reactivity potential using a BLASTp screenemploying parameters optimized for short sequence analysis andpreference for minimal substitution and compositional adjustments asspecificity for the intended target sequence is of utmost importance.Probability values for both On-target and Off-target returned resultsare then analyzed and a composite algorithm-generated value is used todetermine an overall specificity rating. The greater the composite valuethe more specific the target sequence.

Candidate HP-Ag sequences that passed with high specificity and lowoff-target potential (66 sequences) were qualified as HP-Ag (Table 10).

TABLE 10 HP-Ag sequences in AKAP4 Specificity Rating Assessed FoldDifference between Candidate HP sequence Specific Target Qualified(HLA Specificity) SEQ ID NO: and Non-Target HP-Ag? SIDDLSFYV (A2) 1191.59E+03 Yes RLSSLVIQM (A2, A3, B15) 121 1.28E+03 Yes MMVSLMKTL (A2, B8)123 8.40E+02 Yes KMDMSNIVL (A2) 127 2.50E+03 Yes VLMLIQKLL (A2, A24, B8)128 1.14E+03 Yes YQDSLGHEV (A2) 129 5.63E+02 Yes LIDSCMKNL (A2) 1343.94E+03 Yes NLHNITGVL (A2, B8) 135 2.65E+03 Yes IMEAMLKRL (A2) 1367.30E+02 Yes MLKRLVSAL (A2, B8, B15) 137 1.52E+03 Yes KINASTDSL (A2) 1388.90E+02 Yes DMSNIVLML (A2) 141 2.90E+03 Yes IVLMLIQKL (A2) 142 5.00E+03Yes LLNENPFKC (A2) 143 1.63E+03 Yes FIDKLVESV (A2) 144 7.00E+02 YesKLVESVMKL (A2, A3, B15) 145 1.73E+03 Yes QLQAVLQWI (A2) 147 2.03E+03 YesFMGDKDGQL (A2, B8) 148 3.33E+03 Yes KLPQVSAKA (A2) 149 2.55E+02 YesKAAEKGYSV (A2, B8) 150 9.00E+02 Yes SLVIQMAHK (A3, A11) 153 3.80E+03 YesSICPSPGNK (A3, A11) 154 2.50E+03 Yes FLYSELSNK (A3, A11) 155 6.75E+02Yes KEFADSISK (A3) 157 5.50E+02 Yes SISKGLMVY (A3, B15) 158 1.75E+02 YesTLKVHSSGK (A3, A11) 160 1.62E+03 Yes HLTQQTKGK (A3) 165 3.83E+03 YesKCGGGQSAK (A3) 166 1.42E+03 Yes NIVLMLIQK (A3, A11) 167 1.90E+03 YesKLLNENPFK (A3, A11) 168 1.74E+03 Yes KLCLIMAKY (A3, B15) 169 9.50E+03Yes SQFNVPMLY (A3, A11, A15) 170 1.60E+04 Yes FYVNRLSSL (A24, B8) 1721.19E+03 Yes KYSNDGAAL (A24) 173 1.37E+03 Yes QFNVPLMYF (A24) 1741.88E+04 Yes IQMAHKEIK (A11) 175 5.50E+03 Yes ISPRTPASK (A3, A11) 1765.93E+02 Yes KQMSQRESK (A3, A11) 177 1.37E+03 Yes MAQSTQYEK (A11) 1791.37E+03 Yes ASMSNRSDK (A3, A11) 180 2.12E+03 Yes PPSTQRAVI (A24, B7)185 5.20E+02 Yes KPIPASVVL (A24, B7) 187 5.79E+02 YesDPKCRNQSK (A24, B7, B8) 188 7.50E+03 Yes MPQNYQDSL (A24, B7, B8) 1903.84E+03 Yes CSIDDLSFY (B15) 192 4.56E+03 Yes ETKSQSLSY (B15) 1934.89E+03 Yes NQSLEFSTM (B15) 195 4.56E+03 Yes GMKQANGQF (B15) 1965.16E+02 Yes PIPASVVLK (A3, A11) 275 9.68E+02 Yes ELDCTSGMK (A3) 2819.83E+03 Yes QANGQFIDK (A3, A11) 282 6.75E+03 Yes QCSTNSLQK (A3, A11)283 2.98E+03 Yes RQPDEAVGK (A3, A11) 284 1.84E+03 Yes YSELSNKSK (A11)285 9.14E+02 Yes SDMMVSLMK (A11) 286 1.70E+03 Yes TDIMEAMLK (A11) 2876.44E+02 Yes FSTMKAEMK (A11) 288 5.73E+03 Yes GNSCKVATK (A11) 2891.19E+03 Yes EVMKFAKER (A11) 290 8.35E+03 Yes APPAKPPST (A24, B7) 2921.96E+02 Yes MNRPQNLRL (B8) 294 2.49E+03 Yes NLRLEMTAA (B8) 295 2.24E+03Yes DLSFYVNRL (B8) 296 2.20E+03 Yes KLEGKSKCL (B8) 297 1.19E+03 YesEAMLKRLVS (B8) 299 8.92E+02 Yes GVLMTDSDF (B15) 302 1.69E+03 Yes

Example 7 The Derivation of HP-Ag Peptides Homologous to LUZP4(HOM-TES-85) Sequences Expressed in Cancers

The potential of LUZP4 (leuzine zipper protein 4) as an HP-TP wasevaluated using curated literature research as well as data from proteinand genome databases. LUZP4 is a cancer testis antigen that wasidentified by screening a cDNA bank enriched for testis-specifictranscripts with seminoma patient serum (Türeci et al. Ongogene21(24):3879-88 (2002)). LUZP4 is a novel member of the leucine zipperprotein family, which is involved in DNA binding and gene transcription.

Step 1. Qualification of LUZP4 as an HP-TP or Aux-TP

A. TP Frequency

LUZP4 is expressed in a number of cancers including: primary breastcancer (47%, Mischo et al. Int J Cancer 118(3):696 (2006)) liver (19%,Lou et al. Cancer Immun 2:11 (2002)), malignant melanoma (36%), gliomas(35%), cancers (32%), seminomas (31%), lung cancer (28%), liver (19%,Lou), colorectal tumors (9.5%) (Türeci et al. Ongogene 21(24):3879-88(2002)) and Head and Neck Squamous Cell Carcinoma (HNSCC, 4%,Atanackovic et al. Cancer Biol Ther 5(9):1218 (2006)). The level ofexpression of LUZP4 in a wide variety of cancers qualifies it as a TP inregard to frequency.

B. TP Specificity

HOM-TES-85, a cancer testis antigen, is tightly silenced in normaltissues except for testis as determined by RT-PCR and Northern blothybridization studies (Türeci et al. Ongogene 21(24):3879-88 (2002)). Inaddition, resting and activated peripheral blood mononuclear cells donot express LUZP4 indicating that it does not represent a physiologicalproliferation antigen. The lack of LUZP4 expression in normal tissuewhile frequently activated in a number of different cancers gaveHOM-TES-85 a positive specificity value.

C. TP Functional Connectivity

LUZP4 is a cancer testis antigen and a member of the family of leucinezipper proteins, which is involved in RNA export, DNA binding and genetranscription. Studies reveal that LUZP4 localizes to the nucleus whereit could impact the spliceosome or alternatively part of thetranscriptosome in tumor cells (Türeci et al. Ongogene 21(24):3879-88(2002)). Studies by Viphakone et al. (Viphakone et al. Nucleic Acids Res43(4):2353 (2015)) indicate that LUZP4 has two regions that are involvedin mRNA binding. LUZP4 can act as a novel mRNA export adaptor for theTREX export pathway. The TREX complex consists of multiple proteinsthat, together, are a major mRNA export pathway that links transcriptionelongation to mRNA transport from the nucleus to the cytoplasm. Exportof mRNA is often dysregulated in cancer and there is a close linkbetween packaging and export of mRNA and genome stability. For example,the TREX complex is highly expressed in breast cancers and is believedto drive aggressive breast cancer, impacting both tumor size andmetastatic state (Guo et al. Cancer Res. 65:3011 (2015)). LUZP4 enhancesRNA binding activity of the RNA binding domain of nuclear RNA exportfactor 1 (Nxf1) enhancing its binding activity. Nxf1 works inconjunction with another TREX export factor Alyref. LUZP4 is believed tocompete with the normal export factor Alyref.

Another consideration is possible transcriptional function of theleucine zipper region of LUZP4. The leucine zipper region of LUZP4 showsan atypical amphipathy with clusters of hydrophobic residues exclusivelyshared by N-Myc proto-oncogene. Sequence analysis of the zipper regionsuggests a means for involvement of LUZP4 in transcriptional processes.The leucine zipper region of Myc proteins determines sequence specificDNA binding and is essential for myc biology as a cancer driver. Giventhe similarities between the leucine zipper region of N-Myc and LUZP4,it is likely the LUZP4 leucine zipper region can fulfill a similarfunction when abnormally expressed.

LUZP4 is highly expressed in melanoma where it is required for growth ofmelanoma in vitro (Viphakone et al. Nucleic Acids Res 43(4):2353(2015)). In LUZP4 expressing multiple myeloma cell lines, LUZP4knockdown eliminates the colony forming ability of the stem cell-likeside population and their drug resistant properties (Wen et al. Br JHaematol 166:711 (2014)).

The aberrant expression of LUZP4, its potential to impactcancer-associated alterations of transcriptional or post-transcriptionalprocesses, and demonstrated dependence on its expression for qualitiesassociated with Crc qualifies it as a HP-TP antigen.

Step 2. Identification of Candidate HP-Ag Sequences

The LUZP4 sequence used was:

MASFRKLTLSEKVPPNHPSRKKVNFLDMSLDDIIIYKELEGTNAEEEKNKRQNHSKKESPSRQQSKAHRHRHRRGYSRCRSNSEEGNHDKKPSQKPSGFKSGQHPLNGQPLIEQEKCSDNYEAQAEKNQGQSEGNQHQSEGNPDKSEESQGQPEENHHSERSRNHLERSLSQSDRSQGQLKRHHPQYERSHGQYKRSHGQSERSHGHSERSHGHSERSHGHSERSHGHSKRSRSQGDLVDTQSDLIATQRDLIATQKDLIATQRDLIATQRDLIVTQRDLVATERDLINQSGRSH GQSERHQRYSTGKNTITTA total of 313 overlapping 9 amino acid sequences were analyzed for eachHLA type shown. The peptides were evaluated for HLA alleles: A2, A3,A11, A24, B7, B8 and B15.

TABLE 11  Candidate HP-Ag LUZP4 sequences with their HLA specificity HLACore 9mer SEQ Target Specificity sequence ID NO: LUZP4 A2 SLDDIIIYK 310LUZP4 A2 IIYKELEGT 311 LUZP4 A2 KVNFLDMSL 312 LUZP4 A2 FLDMSLDDI 313LUZP4 A2 LIVTQRDLV 314 LUZP4 A3 KVPPNHPSR 315 LUZP4 A3 SLDDIIIYK 310LUZP4 A3 QLKRHHPQY 316 LUZP4 A11 KVPPNHPSR 315 LUZP4 A11 SLDDIIIYK 310LUZP4 A11 NSEEGNHDK 317 LUZP4 A11 PSQKPSGFK 318 LUZP4 A11 GQPLIEQEK 319LUZP4 A11 QSDLIATQR 320 LUZP4 A24 RYSTGKNTI 321 LUZP4 137 MASFRKLTL 322LUZP4 137 HPSRKKVNF 323 LUZP4 B7 SPSRQQSKA 324 LUZP4 B7 KPSQKPSGF 325LUZP4 B7 HPLNGQPLI 326 LUZP4 B8 MASFRKLTL 322 LUZP4 B8 HPSRKKVNE 323LUZP4 B8 PSRKKVNFL 327 LUZP4 B8 RKKVNFLDM 328 LUZP4 B8 KPSQKPSGF 325LUZP4 B8 GFKSGQHPL 329 LUZP4 B8 QLKRHHPQY 316 LUZP4 B8 IATQRDLIV 330LUZP4 B15 RQQSKAHRH 331 LUZP4 B15 EQEKCSDNY 332 LUZP4 B15 QLKRHHPQY 316LUZP4 B15 GQSERSHGH 333 LUZP4 B15 TQRDLIVTQ 334 LUZP4 B15 TQRDLIVTQ 335LUZP4 B15 TQRDLVATE 336 LUZP4 B15 GQSERHQRY 337

Step 3. Screen of HP-Ag Specificity and Off-Target Potential

The selected peptide sequences were then screened for peptidespecificity and off target reactivity potential using a BLASTp screenemploying parameters optimized for short sequence analysis andpreference for minimal substitution and compositional adjustments asspecificity for the intended target sequence is of utmost importance.Probability values for both On-target and Off-target returned resultsare then analyzed and a composite algorithm-generated value is used todetermine an overall specificity rating. The greater the composite valuethe more specific the target sequence.

TABLE 12  HP-Ag sequences that passed with highspecificity and low off-target potential  and qualified as HP-Ag.  Assessed Fold Difference between Candidate HP SEQ Specific sequence IDTarget and Qualified (HLA Specificity) NO: Non-Target HP-Ag?SLDDIIIYK (A2, A3, 310 1.02E+03 Yes A11) IIYKELEGT (A2) 311 2.45E+03 YesKVNFLDMSL (A2) 312 2.36E+03 Yes FLDMSLDDI (A2) 313 9.45E+02 YesLIVTQRDLV (A2) 314 9.55E+02 Yes KVPPNHPSR (A3, A11) 315 2.07E+03 YesQLKRHHPQY (A3, B8, 316 1.13E+04 Yes B15) NSEEGNHDK (A11) 317 4.10E+03Yes PSQKPSGFK (A11) 318 1.89E+03 Yes GQPLIEQEK (A11) 319 1.78E+03 YesQSDLIATQR (A11) 320 1.25E+03 Yes RYSTGKNTI (A24) 321 3.05E+03 YesMASFRKLTL (B7, B8) 322 2.38E+03 Yes HPSRKKVNF (B7, B8) 323 2.53E+03 YesSPSRQQSKA (B7) 324 1.03E+03 Yes KPSQKPSGF (B7, B8) 325 9.30E+02 YesHPLNGQPLI (B7) 326 1.29E+03 Yes PSRKKVNFL (B8) 327 1.64E+03 YesRKKVNFLDM (B8) 328 9.73E+03 Yes GFKSGQHPL (B8) 329 5.52E+03 YesIATQRDLIV (B8) 330 1.32E+03 Yes RQQSKAHRH (B15) 331 7.54E+03 YesEQEKCSDNY (B15) 332 9.30E+03 Yes GQSERSHGH (B15) 333 3.08E+03 YesTQRDLIATQ (B15) 334 2.29E+03 Yes TQRDLIVTQ (B15) 335 2.71E+03 YesTQRDLVATE (B15) 336 1.20E+03 Yes GQSERHQRY (B15) 337 4.77E+03 YesMSLDDIIIY (B15) 338 4.35E+03 Yes

Example 8 The Derivation of HP-Ag Peptides Homologous to the ETV6-NTRK3Sequences Expressed in Cancers

The potential of ETV6-NTRK3 as an HP-TP was evaluated using curatedliterature research as well as data from protein and genome databases.ETV6-NTRK3 is a translocation shared by several rare cancers: secretorycarcinoma of the breast, mammary analogue secretory carcinoma of thesalivary glands (MASC), infantile fibrosarcoma and congenitalmesoblastic nephroma. With the exception of MASC, these cancers areprimarily cancers of infants, children, and young adults. The primarymodality used to treat ETV6-NTRK3 fusion cancers is surgery however thiscan result in amputations and other disfigurement, for example,mastectomy in a child as young as 3 years old with secretory breastcarcinoma (Euhus et al. Cancer Cell 2:347 (2002)) or amputation of alimb to remove infantile fibrosarcoma. Axial congenital fibrosarcomasare considered more aggressive with a recurrence rate as high as 33%(Blocker et al. J Pediatr Surg 22:665 (1987)) with metastases occurringin 13.5% without further therapy beyond surgery. Therefore, furthertreatment is indicated for patients where complete surgical removal isnot possible. Although radiation and chemotherapy are used with goodoverall survival, the use of toxic chemotherapy on young infants couldhave life-long effects. Survivors require close follow-up as sideeffects can occur months to years after the therapy. A safe, targeted Tcell therapy would avoid the serious consequences of current treatmentoptions.

Step 1. Qualification of ETV6-NTRK3 as an HP-TP or Aux-TP

A. TP Frequency

ETV6-NTRK3-driven cancer is rare but present in several types of cancer.The fusion protein is present in 0.15% of breast cancers approximately3,500 diagnoses per year. Most of these patients represent secretorybreast carcinoma where ETV6-NTRK3 is expressed in over 90% of thecancers. Secretory breast carcinoma has a distinctive histopathology.Over 90% of MASC tumors are caused by the ETV6-NTRK3 fusion protein.However MASC represents only about 29 cases of head and neck cancer peryear in the US. ETV6-NTRK3 is expressed in two congenital cancers:infantile or congenital fibrosarcoma and congenital mesoblasticnephroma, which are considered closely related cancers (Adem et al. ModPathol 14:1246 (2001)). Childhood soft tissue sarcomas represent 1% ofall newly diagnosed cancers (Dana Farber Cancer Institute) or anestimated 16,600 cases per year. Congenital fibrosarcomas representapproximately 10% of childhood soft tissue sarcomas (an estimated 1,660cases), commonly located in the extremities (71%) (Blocker et al. JPediatr Surg 22:665 (1987). Twenty-nine percent of congenitalfibrosarcomas are axial where surgical removal is not always possible(Grier et al. Cancer 56:1507 (1985); Blocker et al. J Pediatr Surg22:665 (1987). Infantile or congenital fibrosarcoma and congenitalmesoblastic nephromas are distinguished from other soft tissuefibrosarcomas by the young age of the patient (diagnosed at birth to thefirst 3 months of life). In MASC, ETV6-NTRK3 cancers also havedistinctive histopathology making genetic screening confirmative ratherthan needed for primary diagnosis (Skalova, Head and Neck Pathology7:530 (2013)). Therefore, it is possible to identify patients with MASCbased on presentation and histopathology. Although an HCP therapy wouldhelp patients with all types of ETV6-NTKR3-positive cancers, thefeasibility of ETV6-NTRK3 as an HP-TP is primarily driven by theincidence and ability to identify and reach patients with secretorycarcinoma of the breast, further supported by the congenital cancers.

B. TP Specificity

The ETV6 (ets variant 6) is an ETS family transcriptional repressorexpressed in many normal tissues including lung, colon, heart andsalivary gland (see web-based Proteomics DB,). The native protein playsa role in hematopoiesis. It, in itself is not specific to cancer andtherefore not a target for CTI therapy. NTRK3 (neurotrophic tyrosinekinase, receptor, type 3) protein is reported in the normal brain andretina (Proteomics DB,). The normal protein is not specific to cancerand thus not a target for CTL therapy. The fusion of ETV6 and NTRK3result in unique sequences within the junctional region that arespecific for ETV6-NTRK3, an oncogenic protein present only in cancer.

C. TP Functional Connectivity

NTRK3 is a membrane-bound receptor that upon binding of neurotropin,phosphorylates itself and the RAS-MAP kinase (MAPK) mitogenic pathwayactivating cyclin D1 and the phosphatidyl inositol-3-kinase (PI3K)-AKTcell survival pathway. Fusion of ETV6 with NTRK3 creates a potentprotein tyrosine kinase leading to constitutive activation of the twoNTRK3-mediated pathways. Both are required for the transforming abilityof ETV6-NTRK3 (Tognon et al. Cancer Research 61:8909 (2001)) causingaberrant cell cycle progression, disrupting the balance between thisprogression and apoptosis. Expression of ETV60NTRK3 has been shown to bethe primary event in secretory breast carcinoma evidenced by theretroviral transfer of the fusion protein into murine mammary glandsgiving rise to secretory breast carconima (Tognon et al. Cancer Cell2:367 (2002). Li et al. (Li et al. Cancer Cell 12:542 (2007)) found thatactivation of the fusion oncogene in mice by Wap-Cre leads to 100%penetration of multifocal, malignant breast cancer through activation ofactivator protein 1 (AP1) transcription factor complex. The target ofthis action was the bipotent luminal progenitor cells of the mammarygland, supporting a Crc context. This evidence qualified the functionalconnectivity of ETV6-NTRK3.

ETV6-NTRK3 met the three criteria and therefore qualified as an HP-TP.

Step 2. Identification of Candidate HP-Ag Sequences

The ETV6-NTRK3 sequences used to identify high probability candidateHP-Ag were:

(SEQ ID NO: 339) VSPPEEHAMPIGRIADVQHIKRRDIVLKRELGEGAFGKVFLA  and(SEQ ID NO: 340) LDAGPDTVVIGMTRIPVIENPQYFRQGHNCHKPDTYVQHIKRRDIVLKRELGEGAF Overlapping 9 amino acid sequences were analyzed for each HLA typeshown. The peptides were evaluated for HLA alleles: A2, A3, A11, A24,B7, B8 and B15.

TABLE 13  Candidate HP-Ag sequences in ETV6-NTRK3with their HLA specificity HLA Core 9mer   Target Specificity sequenceSEQ ID NO: ETV6-NTRK3 A2 GAFGKVFLA 341 A2 VIGMTRIPV 342 A3, A11RIADVQHIK 343 A3, A11 ELGEGAFGK 344 A3, A11 VIENPQYFR 345 A11 DTYVQHIKR346 A24, B8 IGMTRIPVI 347 A24 PVIENPQYF 348 B7 PPEEHAMPI 349 B7, B8MPIGRIADV 350 B7 KPDTYVQHI 351 B7, B8 HIKRRDIVL 352 B8 VQHIKRRDI 353

Step 3. Screen of HP-Ag Specificity and Off-Target Potential

The selected peptide sequences were then screened for peptidespecificity and off target reactivity potential using a BLASTp screenemploying parameters optimized for short sequence analysis andpreference for minimal substitution and compositional adjustments asspecificity for the intended target sequence is of utmost importance.Probability values for both On-target and Off-target returned resultsare then analyzed and a composite algorithm-generated value is used todetermine an overall specificity rating. The greater the composite valuethe more specific the target sequence.

TABLE 14  HP-Ag sequences that passed with highspecifity and low off-target  potential were qualified as HP-Ag.Specificity Rating (Fold Difference Candidate HP SEQ between Specificsequence ID Target and Qualified (HLA Specificity) NO: Non-Target)HP-Ag? GAFGKVFLA (A2) 341 1.49e+01 No VIGMTRIPV (A2) 342 4.49E+03 YesVIENPQYFR  345 5.81E+03 Yes (A3, A11) DTYVQHIKR (A11) 346 6.53E+03 YesIGMTRIPVI  347 7.19E+02 Yes (A24, B8) PVIENPQYF (A24) 348 3.29E+03 YesPPEEHAMPI (B7) 349 5.63E+03 Yes KPDTYVQHI (B7) 351 2.35E+03 YesHIKRRDIVL  352 5.59E+03 Yes (B7, B8)

Example 9 HP-Ag Peptides with Homology to Sequences within the FusionRegion Shared by SS18-SSX1 and SS18-SSX2

Synovial sarcomas are high-grade spindle cell tumors accounting for5-10% of all soft tissue sarcomas (Ladanyi et al. Oncogene 20:5755(2001); SEER Cancer Statistics Revew 2008-2012, NCI). Over 90% of thesecancers possess an SS18-SSX (synovial sarcoma X chromosome breakpoint)translocation. There are three histologic subtypes: biphasic, monophasicand poorly differentiated (Ruggiero, Orphanet Encyclopedia, March(2014); Ladanyi et al. Oncogene 20:5755 (2001)). Biphasic tumors containboth epithelial cells with glandular morphology and spindle cells whilemonophasic tumors consist primarily of spindle cells. Poorlydifferentiated tumors have both epithelial and spindle cells with areasof small, pleiopmorphic, cells with numerous mitoses and areas ofnecrosis (Ruggiero, Orphanet Encyclopedia, March (2014)). All subtypesexpress the fusion protein. Synovial sarcoma is more common inadolescents and young adults (Cironi et al., PLoS ONE 4:7904 (2009)). Itis a high-grade cancer currently requiring a multimodal therapy toimprove 5-year survival. Successful surgical removal is the primarytreatment modality. Synovial sarcoma most often metastasizes to thelungs and the outcome for young people with metastatic disease is poor.Both SYT and SSX proteins have the ability to impact transcriptionalregulation. The SSX family members are highly homologous, SSX2 being theprototype. SSX1, 2 and 3 are expressed in multiple cancers (Türeci etal. Int J Cancer 77:19 (2008)). Because of this, others described SSX2(also known as HOM-MEL 40) as a target for T cell therapy even thoughthe function of SSX2 was unknown (Abate-Dega et al., PLoS ONE 9:93321(2014)). In synovial sarcoma the SS18-SSX1 fusion is twice as prevalentas SS18-SSX2 (Ladanyi et al. Oncogene 20:5755 (2001)). Thus, targetingof the SS18-SSX fusions in synovial sarcoma with T cell therapy could bean effective way of ridding young patients of the cancer.

Step 1. Qualification of SS18-SSX1 and SS18-SSX2 as HP-TP or Aux-TP

A. TP Frequency

Synovial sarcoma is rare, representing approximately 5-10% of all softtissue sarcomas. The American Cancer Society estimates that 11,930 newsoft tissue sarcomas will be diagnosed in the US in 2015 and 4,870Americans will die of soft tissue sarcoma. This estimates synovialsarcoma at up to 1,930 new cases and 487 deaths in 2015. Because adiagnosis of synovial sarcoma can be distinguished from other forms ofsarcoma based on primary tumor location and histopathology, and 90% ormore are likely to express the fusion protein, it is feasible to reachthis population within the larger population of soft tissue sarcomaseven though the total number of patients per year is small. Therefore,SS18-SSX fusions meet the criteria of Step 1.

B. TP Specificity

SS18 (synovial sarcoma translocation, chromosome 18) is a ubiquitouslyexpressed normal protein that acts as a transcriptional coactivator. Incontrast, The synovial sarcoma breakpoint proteins SSX1 and SSX2 arehighly homologous cancer-testis antigens. The SSX2 protein is normallyexpressed in the testis and to a lesser extent in the ovaries (Wilhelmet al. Nature 9; 509(7502):582-7 (2014)) and weak expression in thethyroid (Türeci et al. Int. J. Cancer 77:19 (2008)). The fusion proteinis specific to synovial sarcoma and the fusion region offers uniquepeptide sequences for targeting the active fusion protein. Thesesequences will be unique neoantigens relevant to both SS18-SSX1 andSS18-SSX2 in synovial sarcoma. The fusion region meets the requirementfor cancer specificity.

C. TP Functional Connectivity

The fusion protein is an oncogenic transcriptional regulator (Trautmannet al. Oncogene 1-11 (2013)). The fusion puts the expression of SSXunder control of the SYT promoter (Soulez et al. Oncogene 18:2739(1999). Both the SYT and SSX portions of the fusion protein conferoncogenic properties. SYT-SSX1 demonstrates transforming ability in cellculture and nude mouse models by impacting chromatin remodeling.Conditional expression of SS18-SSX in mice can recapitulate manycharacteristics of human synovial sarcoma if expressed in myoblasts, butnot more differentiated myocytes (Cironi et al., 2009, PLoS ONE 4:7904(2009)). This is evidence that the action of SS18-SSX is relevant in aC-RC context. In addition, the ability to target the polycomb complex issignificant to stem cell regulation (Rajasekjar and Begemann Stem Cells25:2498 (2007)). The SSX function of the fusion protein antagonizes thepolycomb complex Bmi1 leading to impairment of Bmi1 function,antagonizing polycomb silencing leading to depression of polycomb targetgenes (Barco et al. PLos One 4:5060 (2009)). Polycomb silencing plays arole in cell fate determination, self-renewal in embryonic and adultstem cells (Rajasekjar and Begemann Stem Cells 25:2498 (2007)). TheSS18-SSX fusion protein induces oncogenic changes in Wnt/beta-cateninsignaling (Trautmann et al. Oncogene 1-11 (2013)) as well as severaladditional members of the Notch signaling pathway (Barco et al. PLos One4:5060 (2009)), promoting cellular transformation. SS18, interacts withglobal chromatin remodeling co-activators (Perani et al. J Biol Chem52:4263 (2005); Cironi et al., 2009, PLoS ONE 4:7904 (2009)). The nativeform of SS18 encodes a nuclear receptor co-activator to activatetranscription (Iwasaki, et al. Endocrinology 146:3892 (2005)). Also, theSS18 portion of the fusion protein both bind to chromatin remodelingfactor hBRM/hSNF2alpha. Studies in vitro and in vivo indicate that Thisaction is essential for tumorigenesis in synovial sarcoma (Nagai et al.Proc Nat Acad Sci. USA 98:3843 (2001)). Binding of the native SS18 tohBRM/hSNF2alpha alone does not lead to transformation. The oncogenicactivity of the SS18 portion of SS18-SSX1 requires the presence of SSX1sequence (Nagai et al. Proc Nat Acad Sci. USA 98:3843 (2001)).

The ability of both the SS18 and the SSX1 and SSX2 portions of thefusion protein to affect chromatin remodeling leading to transformationand depression of key factors in stem cell regulation establish thefunctional connectivity of the fusion protein and its pivotal connectionto the perpetuation of synovial sarcoma.

Step 2. Identification of Candidate HP-Ag Sequences

The SS18-SSX1/SSX2 fusion sequence used was:

QGNDFDNDHNRRIQVEHPQMTFGRLHRIIPKIMPKKPAEDENDSK

The peptides were evaluated for HLA alleles: A2, A3, A11, A24, B7, B8and B15.

TABLE 15 Candidate HP-Ag SS18-SSX1 and SS1S-SSX2sequences with their HLA specificity HLA Core 9 mer SEQ TargetSpecificity sequence ID NO: SS18-SSX1 or 2 A2, B8 MTFGRLHRI 354SS18-SSX1 or 2 A2, A24 RLHRTIPKI 355 SS18-SSX1 or 2 A2 GMVGGGPPA 356SS18-SSX1 or 2 A3, A11 QMTFGRLHR 357 SS18-SSX1 or 2 A11 RIIPKIMPK 358SS18-SSX1 or 2 A11 IIPKIMPKK 359 SS18-SSX1 or 2 A11 QSGPPPPPR 360SS18-SSX1 or 2 A24 TFGRLHRII 361 SS18-SSX1 or 2 B7, B8 HPQMTFGRL 362SS18-SSX1 or 2 B7 MPMGPGGMN 363 SS18-SSX1 or 2 B7 PPPPRSHNM 364SS18-SSX1 or 2 B7 PPRSHNMPS 365

Step 3. Screen of HP-Ag Specificity and Off-Target Potential

The selected peptide sequences were then screened for peptidespecificity and off target reactivity potential using a BLASTp screenemploying parameters optimized for short sequence analysis andpreference for minimal substitution and compositional adjustments asspecificity for the intended target sequence is of utmost importance.Probability values for both On-target and Off-target returned resultsare then analyzed and a composite algorithm-generated value is used todetermine an overall specificity rating. The greater the composite valuethe more specific the target sequence.

TABLE 16  HP-Ag sequences that passed with highspecificity and low off-target potential and qualified as HP-Ag.Specificity Rating (Fold Difference Candidate  SEQ between SpecificHP sequence ID Target and Qualified (HLA Specificity) NO: Non-Target)HP-Ag? RLHRIIPKI (A2, A24) 355 1.14E+03 Yes GMVGGGPPA (A3) 356 1.15E+03Yes TFGRLHRII (A24) 361 3.84E+03 Yes HPQMTFGRL (B7) 362 7.72E+02 YesMPMGPGGMN (B7) 363 7.39E+03 Yes PPPPRSHNM (B7) 364  2.1E+03 YesPPRSHNMPS (B7) 365 1.85E+03 Yes

Example 10 The Derivation of AuxP-Ag Peptides Homologous to LY6KSequences Expressed in Cancers

The potential of LY6K (lymphocyte antigen 6 complex, locus K) as anHP-TP was evaluated using curated literature research as well as datafrom protein and genome databases. LY6K is a cancer-testis antigen thatbelongs to the LY6 superfamily. LY6K shows a high homology to thelow-molecular weight GPI-anchored molecule.

Step 1. Qualification of LY6K as an HP-TP or Aux-TP

A. TP Frequency

LY6K is expressed in 85% of gastric cancers (Ishikawa H et. al. GastricCancer. (1):173-80 (2014)), 88.2% of NSCLC and 95.1% of ESCC (Ishikawa Net. al. Cancer Res. 67(24):11601-11 (2007)). The overexpression of LY6Khas also been documented in a number of cancers including: gingivobuccalcomplex (GBC) cancers (Ambatipudi et. al., Genes Chromosomes Cancer.51(2): 161-173. (2012)), breast cancer (Lee J et. al. Oncol. Rep. 16,1211-1214 (2006)), bladder cancer (Matsuda R. Br. J. Cancer 104, 376-386(2011)), and head and neck squamous cell carcinoma (de Nooij-van Dalen Aet. al. Int J Cancer. March 1; 103(6):768-74 (2003)). LY6K expression in85% of gastric cancers as well as other cancers met the criteria for TPFrequency.

B. TP Specificity

LY6K is considered a cancer testis antigen. There are some discrepanciesin reported protein expression in normal tissues using the availableprotein databases. Proteomics DB reports expression in the rectum and toa lesser extent, the ovaries while the Human Proteome Map from the HumanProteome Project reports no expression in any tissues other than thetestis and ovaries. The Human Protein Atlas, although somewhat lessreliable based on immunohistochemical localization in tumor samples,reports labeling only in the testis. A check of gene expression usingGTex analysis shows very low level to no gene expression in all tissuesbut the testis.

Neo-expression of LY6K in multiple cancers has led to its proposed useas a serologic biomarker for lung and esophageal cancers (Ishikawa etal., Cancer Res 67:11601 (2007)). LY6K peptides are also being tested asa component in multi-peptide cancer vaccines for esophageal cancer (Konoe al. J Translational Medicine 10:141 (2012) and gastric cancer(Ishikawa et al. Gastric Cancer 17:173 (2014); Higashihara et al. Int JOncology 44:662 (2014)). However, to our knowledge, no one has proposedor described the use of LY6K epitopes to design CTL-based therapy. LY6Kwas given a positive specificity value based on 1) Lack of LY6K proteinexpression in normal tissues other than testis and possibly the ovaries,supported by multiple databases, and 2) the fact that it is frequentlynewly expressed in a number of cancers, resulted in a positive value forcancer specificity.

C. TP Functional Connectivity

LY6K is a GPI-anchored protein. In sperm it is associated withtestis-expressed gene 101 (TEX101). Together, these proteins arerequired for sperm migration into the oviduct (Fujihara et al. Biologyof Reproduction 90:60 (2014)). The abnormal action of LY6K is associateda gain of function mutation. It lies in close proximity to other knownoncogenes like MYC. Transfection of bladder cancer cells with LY6Kenhances cell migration, invasion into extracellular matrix (Matrigel)and cell proliferation. Conversely, knock out of LY6K results indecreased ability to migrate and invade Matrigel with reducedproliferation (Matsuda et al. Br. J. Cancer 104; 376 (2011)). This isconsistent with normal actions of LY6K in the enabling of sperm tomigrate into the oviduct. Human LY6K belongs to the LY-6.urokinase-typeplasminogen activator receptor (UPAR) superfamily. The urokinase systemis involved in tissue remodeling and is associated with cancer spreadthrough matrix turnover, ability to invade tissue stroma and migrate,enabled proliferation, apoptosis and angiogenesis (Hildebrand and SchaafInt. J. Oncology 34:15 (2008)). Activating protein-1 (AP-1)transcription factors JunD and Fra-1 induce invasion and metastasis ofbreast cancer cells by increasing LY6K gene expression and theactivation of Raf-1/MEK/ERK signaling pathway and up-regulation ofmatrix metalloproteases. (Kong et al. J Biol Chem 287:38889 (2012)).Therefore, the action of LY6K is to enable tumor growth and metastasisby supporting tissue remodeling and cell invasion. Its actions will bedownstream of the pivotal changes in the cancer that will induce AP-1transcription factors. Therefore LY6K is not an HP-TP but is rather anenabling Aux-TP. Aux-TPs can serve as useful second or companion targetsin an HP-ACT therapy, particularly in advanced cancer with activemetastases.

Step 2. Identification of Candidate HP-Ag Sequences

TABLE 17  Candidate HP-Ag sequences in LY6K with their HLA specificityHLA Core 9mer SEQ ID Target Specificity sequence NO:  LY6K A2 GTMALLALL366 A2 MALLALLLV 367 A2 ALLALLLVV 368 A2 LLVVALPRV 369 A2 KIFPRFFMV 380A2 FMVAKQCSA 370 A2 SMGESCGGL 371 A2 GLWLAILLL 372 A2 WLAILLLLA 396 A2AILLLLASI 374 A2 ILLLLASIA 375 A2 LLLLASIAA 376 A2 LLASIAAGL 377 A2, B8LALLLVVAL 379 A2, B15 FLLEEPMPF 378 A3 LLLVVALPR 381 A3 RVWCHVCER 382 A3KIFPRFFMV 380 A11 LLLVVALPR 381 A11 RVWCHVCER 382 A11 NTFECQNPR 383 A24KWTEPYCVI 384 A24 AAVKIFPRF 385 A24 LWLAILLLL 386 B7 APRADPPWA 387 B7RADPPWAPL 388 B7 PPWAPLGTM 389 B7 WAPLGTMAL 390 B7 APLGTMALL 391 B8CCKIRYCNL 392 B15 CVIAAVKIF 393 B15 AVKIFPRFF 394 B15 KQCSAGCAA 395 B15LLEEPMPFF 396 B15 YLKCCKIRY 397

Step 3. Screen of HP-Ag Specificity and Off-Target Potential

The selected peptide sequences were then screened for peptidespecificity and off target reactivity potential using a BLASTp screenemploying parameters optimized for short sequence analysis andpreference for minimal substitution and compositional adjustments asspecificity for the intended target sequence is of utmost importance.Probability values for both On-target and Off-target returned resultsare then analyzed and a composite algorithm-generated value is used todetermine an overall specificity rating. The greater the composite valuethe more specific the target sequence.

TABLE 18HP-Ag sequences that passed with high specificity and low off-targetpotential and qualified as HP-Ag. Specificity Rating (Fold DifferenceCandidate HP sequence SEQ between Specific Qualified (HLA Specificity)ID NO: Target and Non-Target) HP-Ag? LLVVALPRV (A2) 369 1.05E+03 YesFMVAKQCSA (A2) 370 1.54E+04 Yes SMGESCGGL (A2) 371 2.12E+03 YesGLWLAILLL (A2) 372 7.07E+02 Yes FLLEEPMPF (A2, B15) 378 9.66E+03 YesKIFPRFFMV (A3) 380 2.26E+04 Yes RVWCHVCER (A3, A11) 382 3.96E+04 YesNTFECQNPR (A11) 383 1.34E-04 Yes KWTEPYCVI (A24) 384 4.10E+04 YesAAVKIFPRF (A24) 385 1.78E+03 Yes LWLAILLLL (A24) 386 8.94E+02 YesAPRADPPWA (B7) 387 3.40E+03 Yes RADPPWAPL (B7) 388 9.60E+02 YesPPWAPLGTM (B7) 389 1.21E+04 Yes WAPLGTMAL (B7) 390 3.58E+03 YesCCKIRYCNL (B8) 392 4.02E+04 Yes AVKIFPRFF (B15) 394 3.19E+03 YesKQCSAGCAA (B15) 395 1.76E+03 Yes LLEEPMPFF (B15) 396 1.34E+04 YesYLKCCKIRY (B15) 397 1.49E+04 Yes

Example 11 The Ability of Core High Probability 9Mers of Step 2 toIdentify Suitable Epitopes of Varied Length

Historically, T cell antigens described by others have been of varyinglengths. When working with short protein sequences, such as a relativelyshort fusion region created by a translocation or the unique portion ofa protein that is a member of a large, related family, it is desirableto identify as many specific antigenic High Probability (HP) peptides aspossible. Although a 9 amino acid sequence (9mer) is the naturalsequence length for HLA binding, peptides of 8, 10, and 11 amino acids(8mer, 10mer and 11mer respectively) can also bind the HLA cleft andserve as T cell antigens. However, comprehensive data is scarce forpeptides of lengths beyond the standard 9mer. Therefore we wanted to 1)determine if the HP 9mer core peptides were the best configuration inmost instances and 2) if they would predict feasible alternativepeptides of 8. 1. Or 11 amino acids. We tested the ability of HLA A2core 9mer sequences of the TMPRSS2-ERG fusion region identified by Step2 (Example) to select suitable peptides of differing lengths that couldbe HP cytotoxic T cell antigens. Step 2 had identified 6 HP 9merepitopes within the fusion region out of a possible 212 overlappingpeptide sequences.

Studies were first conducted to determine if the characteristics of anyof the six 9mer peptides would be improved by either subtracting oneamino acid on either end to form an 8mer or adding 1 or 2 amino acids oneither end to form 10mers and 11mers. The resulting peptide sequenceswere analyzed for changes in affinity to HLA-A2 (NetMHC 3.4, Nielsen etal. Protein Sci., 12:1007-17 (2003)) and peptide processing (IEDB,Tenzer et al. Cell Mol Life Sci. 62:1025-1037 (2005)).

In this case, addition or subtraction of amino acids on the C and Nterminal ends resulted in a significant decrease in predicted affinitycompared to the HP 9mer core sequences, with only slight improvements inprocessing in some instances (Table 1). Therefore, targeting the 9mercore is the preferred method to identify T cell antigens.

TABLE 19 Comparison of TMPRSS2-ERG HLA A2 HP 9 mer corepeptides (bold) and associated sequences of differing lengths SEQAffinity Processing ID NO: (Kd, nM) Score WLSQPPAR 398 18192 1.68LSQPPARV 399 21095 1.16 WLSQPPARV  84   161 1.11 DWLSQPPARV 400 240431.13 WLSQPPARVT 401 16544 0.47 QDWLSQPPARV 402 18934 1.1 DWLSQPPARVT 40324991 0.5 WLSQPPARVTI 404  1430 1.64 KMECNPSQ 405 23287 0.92 MECNPSQV406 23020 1.16 KMECNPSQV  89   463 1.19 IKMECNPSQV 407  5912 1.22KMECNPSQVN 408 29989 0.5 TIKMECNPSQV 409 14816 1.22 IKMECNPSQVN 41027187 0.52 KMECNPSQVNG 411 19841 0.45 KMVGSPDT 412  9448 0.44 MVGSPDTV413  2352 1.43 KMVGSPDTV  85    56 1.5 GKMVGSPDTV 414 11012 1.34KMVGSPDTVG 415 16040 0.55 GGKMVGSPDTV 416 25046 1.23 GKMVGSPDTVG 41729682 0.39 KMVGSPDTVGM 418   930 1.5 VIVPADPT 419 11852 0.15 IVPADPTL420  7954 2.06 VIVPADPTL  86  1103 2.15 RVIVPADPTL 421  4482 2.23VIVPADPTLW 422 24055 2 RRVIVPADPTL 423 15915 2.13 RVIVPADPTLW 424 239082.08 VIVPADPTLWS 425 14849 0.37 GLPDVNIL 426   960 1.75 LPDVNILL 42722931 1.79 GLPDVNILL  87    14 1.91 YGLPDVNILL 428  1141 1.82 GLPDVNILLF429  2887 2.24 EYGLPDVNILL 430 22786 2.01 YGLPDVNILLF 431  8393 2.15GLPDVNILLFQ 432  5778 1.03 ILLSHLHY 433  4623 2.47 LLSHLHYL 434   1791.82 ILLSHLHYL  88     3 1.77 DILLSHLHYL 435  2148 1.8 ILLSHLHYLR 436 1732 1.66 ADILLSHLHYL 437  9940 1.82 DILLSHLHYLR 438 28058 1.69ILLSHLHYLRE 439 11054 0.31

Studies were then conducted to examine whether HP 9mer core peptidesderived from Step 2, would identify HP epitopes of varied lengths. Wesurveyed the fusion region identified in Example using NetMHC 4.0(Andreatta et al. Bioinformatics (2015)—epublished ahead of print Nov.13, 2015), which reports a core sequence based on sequence alignment fora given allele, rank and N terminal binding for peptides of 8-11 aminoacids, trained on IEDB MHC Class I affinity measurements. We found that9mer sequences identified for HLA A2 were contained in the 8mer (1 of2), 10mer (2 of 3) and 11mer (2 of 2) peptides identified by NetMHC 4.0using the authors' preset parameters. One 8mer, FIFPNTSV (SEQ ID NO:440)and one 10mer, YLRETPLPHL (SEQ ID NO:441), powered by calculatedaffinity, did not contain an HP 9mer core peptide. Processing andaffinity scores for FIFPNTSV (SEQ ID NO:440) and YLRETPLPHL SEQ IDNO:441) fit within the range exhibited by the HP-9mer core peptides,qualified based on the comprehensive set of Step 2 parameters.Therefore, although data is scarce for peptides of varied lengths beyond9 amino acids, comparison with the 9 mer core values can be used tocorroborate the utility of epitopes of varying lengths. Both FIFPNTSV(SEQ ID NO:440) and YLRETPLPHL SEQ ID NO:441) would likely perform asadditional HP epitopes for the TMPRSS2-ERG fusion region as they comparefavorably to the range established by the six HP 9mer antigens, forexample, in processing and affinity

TABLE 20 Comparison of sample values between 9 mercore sequences and epitopes of varying length identified by NetMHC 4.0SEQ Processing Affinity HP core sequences ID NO: Score (Kd, nM)WLSQPPARV  84 1.11  161 KMECNPSQV  89 1.19  463 KMVGSPDTV  85 1.5   56VIVPADPTL  86 2.15 1103 GLPDVNILL  87 1.91   14 ILLSHLHYL  88 1.77    3Sequences identified only by Net MHC 4.0, corroboratedusing 9 mer core data FIFPNTSV 440 1.14  118 YLRETPLPHL 441 1.99   34

The ability of the 9mer core to predict epitopes of varying lengths in alonger sequence was tested, AKAP4 consisting of a total of 678overlapping 9 amino acid sequences. We used NetMHC 4.0 under its presetparameters to identify binding peptides for overlapping sequences of8-11 amino acids. As shown in Table 20, core HLA A2 AKAP4 9mersidentified by this method were shared in all but one 10mer sequenceSLAKDLIVSA (SEQ ID NO: 269) identified by NetMHC 4.0 as a peptidecapable of binding HLA A2.

TABLE 21 Comparison of core HLA A2 AKAP4 sequencesidentified by various methods NetMHC 4.0 Step 2 NetMHC 4.0 NetMHC 4.0High affinity Qualified High affinity High affinity 8 mer HP 9 mer core10 mer 11 mer IDDLSFYV SIDDLSFYV CSIDDLSFYV ECSIDDLSFYV (SEQ ID NO: 442)(SEQ ID NO: 119) (SEQ ID NO: 443) (SEQ ID NO: 270) SIDDLSFYVN(SEQ ID NO: 444) GLMVYANQV KGLMVYANQV (SEQ ID NO: 122) (SEQ ID NO: 445)MMVSLMKTL MMVSLMKTLKV (SEQ ID NO: 123) (SEQ ID NO: 306) VLMTDSDFVGVLMTDSDFV LMTDSDFVSAV (SEQ ID NO: 125) (SEQ ID NO: 446)(SEQ ID NO: 307) VLMTDSDFVS (SEQ ID NO: 447) AMLKRLVSA AMLKRLVSAL(SEQ ID NO 126) (SEO ID NO: 137) KMDMSNIVL KMDMSNIVLM (SEQ ID NO: 127)(SEQ ID NO: 448) MDMSNIVLML (SEQ ID NO: 274) FIDKLVESV QFIDKLVESV(SEQ ID NO: 144) (SEQ ID NO: 273) KLVESVMKL DKLVESVMKL (SEQ ID NO: 145)(SEQ ID NO: 272) LLQEVMKFA GLLQEVMKFA (SEQ ID NO: 152) (SEQ ID NO: 305)LLDWLLANL QLLDWLLANL KQLLDWLLANL (SEQ ID NO: 132) (SEQ ID NO: 271)(SEQ ID NO: 308)

Since affinity is only one aspect of an effective T cell antigen, thenovel peptide was qualified by comparing calculable 10mer values to theHP core sequences that identified NetMHC 4.0-positive sequences. Acomparison on processing scores and affinities are provided in Table 21as an example. It should be noted that in this larger sequence, Step 2identified additional 9mers not identified by NetMHC 4.0's presetparameters, creating the possibility of further expanding the pool ofepitope candidates based on a range established using the 9mer corepeptides.

TABLE 22 Comparison of processing scores andaffinities of HP 9 mer core sequences Identifying HP 9 mer SEQProcessing Affinity core sequences ID NO: Score (Kd, nM) SIDDLSFYV 1191.07   3 GLMVYANQV 122 1.22  18 MMVSLMKTL 123 2.17  75 VLMTDSDFV 1251.23   5 AMLKRLVSA 126 1.04  52 KMDMSNIVL 2.07  61 FIDKLVESV 144 1.04 13 KLVESVMKL 145 1.89  10 LLQEVMKFA 152 0.98 121 LLDWLLANL 132 1.8  19Sequence identified only by Net MCH 4.0 affinity prediction SLAKDLIVSA269 1.11  98

The 9 mer core sequences were highly predictive of high affinity T cellantigens having varying numbers of amino acids. Also, the use of HP 9merranges established for HLA-A2 could serve as a metric to corroborate theHP potential of epitopes of varying length where reliable data is stillscarce.

Modifications and variations of the methods and materials describedabove will be obvious to those skilled in the art from the foregoingdetailed description and are intended to come within the scope of theappended claims. References cited herein are specifically incorporatedby reference.

We claim:
 1. A method for identifying T-cell epitopes which target cellscapable of regenerating cancers comprising: (i) identifying highcurative potential tumor protein targets (HP-TP) or auxiliary tumorprotein targets (Aux-TP); (ii) identifying peptide sequences within theprotein sequence of an HP-TP or Aux-TP that have a high probability ofeliciting T cell killing; and (iii) qualifying the sequence specificitybased on the fold difference between the specific target andnon-targets.
 2. The method of claim 1, wherein the HP-TP is identifiedbased on its frequency, specificity and functional connectivity.
 3. Themethod of claim 2, wherein the frequency parameters are selected fromthe group consisting of pattern of expression, clinical and commercialfeasibility (Frequency).
 4. The method of claim 2, wherein specificityis calculated from the HP-TP's its ability to discriminate cancer cellsfrom normal cells and the functional connectivity is based on thecalculated strength of its functional relationship to the cancer'sability to perpetuate itself.
 5. A method of identifying cancerregeneration capable cell©-RC cell-specific T cells, comprising mixingepitopes isolated according to the method of claim 1 with isolated CD8⁺T cells from normal or cancer donors, isolating T cells that react withthe epitope, wherein the reactive T cells comprise T cell receptors thattarget a C-RC-specific antigen.
 6. The method of claim 5, furthercomprising adding between initial lymphocyte isolation and the selectionmethod an in vitro activation protocol selecting for CD8⁺ T cells thatare reactive to appropriately presented peptide antigen in the contextof patient-relevant HLA molecules.
 7. The method of claim 5, furthercomprising cloning the T cell receptors (TCR) from the reactive T cellsto produce a cloned TCR construct.
 8. The method of claim 7, furthercomprising transducing a population of T cells with the cloned TCRconstruct and testing the transduced T cells for their ability torespond to expressed HLA-restricted peptide complex.
 9. The method ofclaim 7, comprising forming a banked TCR panel.
 10. An isolated tumorepitope prepared by the method of claim
 1. 11. A method of treatingcancer in a patient comprising administering to the patient T cellstransduced with an expression vector encoding T cell receptors reactivewith the one or more antigens identified or obtained by assaying acancer biopsy from the patient to determine the presence of one or moreantigens identified using the method of claim
 8. 12. The method of claim8, wherein the T cells are obtained from the patient prior totransduction.
 13. The method of claim 12, further comprising partiallyimmunodepleting the patient prior to administration of the T cells. 14.The method of claim 12, wherein the T cells are transduced to expresscytokine or adjuvant to enhance the T cell response.
 15. The epitopesidentified using the method of claim
 1. 16. The epitopes of claim 15,derived from BRD4-NUT (bromodomain containing 4 protein-nuclear proteinin testis) fusion regions selected from the group consisting of SEQ IDNOs:27, 31-32, 38-39, 41, 44-47, 200-203, 209-211, 213-216, 218-220,222-223, 225-230 and
 234. 17. The epitopes of claim 15, derived from ALKfusion protein comprising a sequence selected from SEQ ID NOs: 56-82,235, 237-245 and 248-249.
 18. The epitopes of claim 15, derived fromtransmembrane protease, serine 2-erythroblast transformation specific(ETS)-related gene) (TMPRSS2-ERG) comprising a sequence selected fromSEQ ID NOs:84, 86-93, 95-98, 100, 102-105, 107, 109-113, 115, 117, 250,251-267 and 268-274.
 19. The epitopes of claim 15 derived from A-kinaseanchoring protein 4) (AKAP4) comprising a sequence selected from SEQ IDNOs:119, 121, 123, 127-129, 134-138, 141-145, 147-150, 153-155, 157-158,160, 165-170, 172, 180, 185, 187-188, 190, 192-193, 195-196, 275,281-290 and
 292. 20. The epitopes of claim 15, derived from leucinezipper protein 4 (LUZP4), comprising a sequence selected from SEQ IDNOs. 310-329.
 21. The epitopes of claim 15, derived from ETS variant6-neurotrophic tyrosine kinase, receptor, type 3 (ETV6-NTRK3) fusion,comprising a sequence selected from SEQ ID NOs: 341-342, 345-349 and351-352.
 22. The epitopes of claim 15 comprises a sequence selected fromSEQ ID NOs: 354-365.
 23. The epitopes of claim 15, derived fromlymphocyte antigen 6 complex, locus K (LY6K), comprising a sequenceselected from SEQ ID NOs: 36-372, 378, 380, 382-390, 392 and 394-397.